Aqueous solubilization of transmembrane peptide sequences with retention of membrane insertion and function.

We recently reported that the peptide C-K4-M2GlyR mimics the action of chloride channels when incorporated into the apical membrane of cultured renal epithelial monolayers. C-K4-M2GlyR is one of a series of peptides that were prepared by the addition of lysine residues to the N- or C-terminus of the M2 transmembrane sequence of the brain glycine receptor. This study addresses how such modifications affect physical properties such as aqueous solubility, aggregation, and secondary structure, as well as the ability of the modified peptides to form channels in epithelial monolayers. A graded improvement in solubility with a concomitant decrease in aggregation in aqueous media was observed for the M2GlyR transmembrane sequences. Increases in short-circuit current (I(SC)) of epithelial monolayers were observed after treatment with some but not all of the peptides. The bioactivity was higher for the more soluble, less aggregated M2GlyR peptides. As described in our previous communication, sensitivity of channel activity to diphenylamine-2-carboxylate, a chloride channel blocker, and bumetanide, an inhibitor of the Na/K/2Cl cotransporter, was used to assess changes in chloride selectivity for the different assembled channel-forming peptides. The unmodified M2GlyR sequence and the modified peptides with less positive charge are more sensitive to these agents than are the more highly charged forms. This study shows that relatively insoluble transmembrane sequences can be modified such that they are easier to purify and deliver in the absence of organic solvents with retention of membrane association, insertion, and assembly.     

NH(2)-terminal modification of a channel-forming peptide increases capacity for epithelial anion secretion

A synthetic, channel-forming peptide, derived from the alpha-subunit of the glycine receptor (M2GlyR), has been synthesized and modified by adding four lysine residues to the NH(2) terminus (N-K(4)-M2GlyR). In Ussing chamber experiments, apical N-K(4)-M2GlyR (250 microM) increased transepithelial short-circuit current (I(sc)) by 7.7 +/- 1.7 and 10.6 +/- 0.9 microA/cm(2) in Madin-Darby canine kidney and T84 cell monolayers, respectively; these values are significantly greater than those previously reported for the same peptide modified by adding the lysines at the COOH terminus (Wallace DP, Tomich JM, Iwamoto T, Henderson K, Grantham JJ, and Sullivan LP. Am J Physiol Cell Physiol 272: C1672-C1679, 1997). N-K(4)-M2GlyR caused a concentration-dependent increase in I(sc) (k([1/2]) = 190 microM) that was potentiated two- to threefold by 1-ethyl-2-benzimidazolinone. N-K(4)-M2GlyR-mediated increases in I(sc) were insensitive to changes in apical cation species. Pharmacological inhibitors of endogenous Cl(-) conductances [glibenclamide, diphenylamine-2-dicarboxylic acid, 5-nitro-2-(3-phenylpropylamino)benzoic acid, 4,4'-dinitrostilben-2,2'-disulfonic acid, indanyloxyacetic acid, and niflumic acid] had little effect on N-K(4)-M2GlyR-mediated I(sc). Whole cell membrane patch voltage-clamp studies revealed an N-K(4)-M2GlyR-induced anion conductance that exhibited modest outward rectification and modest time- and voltage-dependent activation. Planar lipid bilayer studies yielded results indicating that N-K(4)-M2GlyR forms a 50-pS anion conductance with a k([1/2]) for Cl(-) of 290 meq. These results indicate that N-K(4)-M2GlyR forms an anion-selective channel in epithelial monolayers and shows therapeutic potential for the treatment of hyposecretory disorders such as cystic fibrosis.     

Chlorzoxazone or 1-EBIO increases Na(+) absorption across cystic fibrosis airway epithelial cells

Previous studies demonstrated that chlorzoxazone or 1-ethyl-2-benzimidazolinone (1-EBIO) enhances transepithelial Cl(-) secretion by increasing basolateral K(+) conductance (G(K)) (Singh AK, Devor DC, Gerlach AC, Gondor M, Pilewski JM, and Bridges RJ. J Pharmacol Exp Ther 292: 778-787, 2000). Hence these compounds may be useful to treat cystic fibrosis (CF) airway disease. The goal of the present study was to determine whether chlorzoxazone or 1-EBIO altered ion transport across Delta F508-CF transmembrane conductance regulator homozygous CFT1 airway cells. CFT1 monolayers exhibited a basal short-circuit current that was abolished by apical amiloride (inhibition constant 320 nM) as expected for Na(+) absorption. The addition of chlorzoxazone (400 microM) or 1-EBIO (2 mM) increased the amiloride-sensitive I(sc) approximately 2.5-fold. This overlapping specificity may preclude use of these compounds as CF therapeutics. Assaying for changes in the basolateral G(K) with a K(+) gradient plus the pore-forming antibiotic amphotericin B revealed that chlorzoxazone or 1-EBIO evoked an approximately 10-fold increase in clotrimazole-sensitive G(K). In contrast, chlorzoxazone did not alter epithelial Na(+) channel-mediated currents across basolateral-permeabilized monolayers or in Xenopus oocytes. These data further suggest that alterations in basolateral G(K) alone can modulate epithelial Na(+) transport.     

Evidence for basolateral Cl- channels as modulators of apical Cl- secretion in pulmonary epithelia of Xenopus laevis

Pulmonary epithelia of air-breathing vertebrates are covered by a thin, fluid layer that is essential for immune defense and gas diffusion. The composition of this layer is maintained by ion transport mechanisms, including Cl(-) transport. The present study focuses on the function of basolateral Cl(-) channels in Xenopus pulmonary epithelia, since knowledge concerning this issue is limited. Therefore, Ussing chamber measurements were performed, and transepithelial short-circuit currents (I(SC)) were monitored. Basolateral application of the Cl(-) channel inhibitor N-phenylanthranilic acid (DPC) resulted in an increase of the I(SC), indicating a DPC-sensitive Cl(-) conductance. This observation was confirmed in experiments using an apical-to-basolateral Cl(-) gradient, with and without nystatin (apical side) to permeabilize the epithelia as well as by establishing an iodide gradient. The DPC-sensitive Cl(-) conductance was influenced by procedures interfering with apical Cl(-) secretion. For example, the effect of forskolin was increased when basolateral Cl(-) channels were blocked by the simultaneous application of DPC. Activation of apical Cl(-) secretion by forskolin/IBMX and subsequent DPC application resulted in a significantly reduced DPC effect. Accordingly, DPC led to an increased apical Cl(-) secretion estimated by an increased 5-nitro-2-(3-phenylpropylamino)benzoic acid-sensitive I(SC). Furthermore, inhibition of basolateral anion exchangers responsible for Cl(-) uptake resulted in a decreased DPC-sensitive current. Taken together, we have evidence concerning the function of basolateral Cl(-) channels in Xenopus pulmonary epithelium and that these channels play a significant role in mediating apical Cl(-) secretion involving a novel Cl(-) recycling mechanism across the basolateral membrane.     

Electrolyte Transport in the Mouse Trachea: No Evidence for a Contribution of Luminal K+ Conductance

Recent studies on frog skin acini have challenged the question whether Cl(-) secretion or Na(+) absorption in the airways is driven by luminal K(+) channels in series to a basolateral K(+) conductance. We examined the possible role of luminal K(+) channels in electrolyte transport in mouse trachea in Ussing-chamber experiments. Tracheas of both normal and CFTR (-/-) mice showed a dominant amiloride-sensitive Na+ absorption under both, control conditions and after cAMP-dependent stimulation. The lumen-negative transepithelial voltage was enhanced after application of IBMX and forskolin and Cl(-) secretion was activated. Electrolyte secretion induced by IBMX and forskolin was inhibited by luminal glibenclamide and the blocker of basolateral Na(+2)Cl(-)K(+) cotransporter azosemide. Similarly, the compound 293B, a blocker of basolateral KCNQ1/KCNE3 K(+) channels effectively blocked Cl(-) secretion when applied to either the luminal or basolateral side of the epithelium. RT-PCR analysis suggested expression of additional K(+) channels in tracheal epithelial cells such as Slo1 and Kir6.2. However, we did not detect any functional evidence for expression of luminal K(+) channels in mouse airways, using luminal 293B, clotrimazole and Ba(2+) or different K(+) channel toxins such as charybdotoxin, apamin and a-dendrotoxin. Thus, the present study demonstrates Cl(-) secretion in mouse airways, which depends on basolateral Na(+2)Cl(-)K(+) cotransport and luminal CFTR and non-CFTR Cl(-) channels. Cl(-) secretion is maintained by the activity of basolateral K(+) channels, while no clear evidence was found for the presence of a luminal K(+) conductance.     

Cyclic AMP-induced K⁺ secretion occurs independently of Cl⁻ secretion in rat distal colon

cAMP induces both active Cl(-) and active K(+) secretion in mammalian colon. It is generally assumed that a mechanism for K(+) exit is essential to maintain cells in the hyperpolarized state, thus favoring a sustained Cl(-) secretion. Both Kcnn4c and Kcnma1 channels are located in colon, and this study addressed the questions of whether Kcnn4c and/or Kcnma1 channels mediate cAMP-induced K(+) secretion and whether cAMP-induced K(+) secretion provides the driving force for Cl(-) secretion. Forskolin (FSK)-enhanced short-circuit current (indicator of net electrogenic ion transport) and K(+) fluxes were measured simultaneously in colonic mucosa under voltage-clamp conditions. Mucosal Na(+) orthovanadate (P-type ATPase inhibitor) inhibited active K(+) absorption normally present in rat distal colon. In the presence of mucosal Na(+) orthovanadate, serosal FSK induced both K(+) and Cl(-) secretion. FSK-induced K(+) secretion was 1) not inhibited by either mucosal or serosal 1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole (TRAM-34; a Kcnn4 channel blocker), 2) inhibited (92%) by mucosal iberiotoxin (Kcnma1 channel blocker), and 3) not affected by mucosal cystic fibrosis transmembrane conductance regulator inhibitor (CFTR(inh)-172). By contrast, FSK-induced Cl(-) secretion was 1) completely inhibited by serosal TRAM-34, 2) not inhibited by either mucosal or serosal iberiotoxin, and 3) completely inhibited by mucosal CFTR(inh)-172. These results indicate that cAMP-induced colonic K(+) secretion is mediated via Kcnma1 channels located in the apical membrane and most likely contributes to stool K(+) losses in secretory diarrhea. On the other hand, cAMP-induced colonic Cl(-) secretion requires the activity of Kcnn4b channels located in the basolateral membrane and is not dependent on the concurrent activation of apical Kcnma1 channels.     

A synthetic peptide based on a glycine-gated chloride channel induces a novel chloride conductance in isolated epithelial cells

CK(4)-M2GlyR, an aqueous soluble peptide derived from the transmembrane M2 segment of the glycine-gated Cl(-) channel found in postsynaptic membranes of the central nervous system, has previously been shown to increase transepithelial Cl(-) and fluid secretion of epithelial monolayers. The goal of this study was to determine whether CK(4)-M2GlyR exerts these effects via formation of a novel chloride conductance pathway, modulation of endogenous chloride channel activity, or a combination of these effects. Ionic currents were recorded from isolated epithelial cells before and after treatment with the peptide using the whole-cell configuration of the patch-clamp technique. CK(4)-M2GlyR increased whole-cell Cl(-) currents in all epithelial cell lines that were studied, including: Madin-Darby canine kidney cells, a human colonic epithelial cell line (T84), and airway epithelial cells derived from a human cystic fibrosis patient (IB3-1). No evidence was found for modulation of endogenous Cl(-) channels by CK(4)-M2GlyR based on both the electrophysiological properties of the observed currents and the pharmacological profile of the CK(4)-M2GlyR-induced current. These results suggest that CK(4)-M2GlyR increases Cl(-) permeability in epithelial cells directly, by forming a distinct conduction pathway in cell membranes.     

Na(+), Cl(-), Ca(2+) and Zn(2+) transport by fish gills: retrospective review and prospective synthesis

The secondary active Cl(-) secretion in seawater (SW) teleost fish gills and elasmobranch rectal gland involves basolateral Na(+),K(+)-ATPase and NKCC, apical membrane CFTR anion channels, and a paracellular Na(+)-selective conductance. In freshwater (FW) teleost gill, the mechanism of NaCl uptake is more controversial and involves apical V-type H(+)-ATPase linked to an apical Na(+) channel, apical Cl(-)-HCO-3 exchange and basolateral Na(+),K(+)-ATPase. Ca(2+) uptake (in FW and SW) is via Ca(2+) channels in the apical membrane and Ca(2+)-ATPase in the basolateral membrane. Mainly this transport occurs in mitochondria rich (MR) chloride cells, but there is a role for the pavement cells also. Future research will likely expand in two major directions, molded by methodology: first in physiological genomics of all the transporters, including their expression, trafficking, operation, and regulation at the molecular level, and second in biotelemetry to examine multivariable components in behavioral physiological ecology, thus widening the integration of physiology from the molecular to the environmental levels while deepening understanding at all levels.     

Apical Ca2+-activated potassium channels in mouse parotid acinar cells

Ca(2+) activation of Cl and K channels is a key event underlying stimulated fluid secretion from parotid salivary glands. Cl channels are exclusively present on the apical plasma membrane (PM), whereas the localization of K channels has not been established. Mathematical models have suggested that localization of some K channels to the apical PM is optimum for fluid secretion. A combination of whole cell electrophysiology and temporally resolved digital imaging with local manipulation of intracellular [Ca(2+)] was used to investigate if Ca(2+)-activated K channels are present in the apical PM of parotid acinar cells. Initial experiments established Ca(2+)-buffering conditions that produced brief, localized increases in [Ca(2+)] after focal laser photolysis of caged Ca(2+). Conditions were used to isolate K(+) and Cl(-) conductances. Photolysis at the apical PM resulted in a robust increase in K(+) and Cl(-) currents. A localized reduction in [Ca(2+)] at the apical PM after photolysis of Diazo-2, a caged Ca(2+) chelator, resulted in a decrease in both K(+) and Cl(-) currents. The K(+) currents evoked by apical photolysis were partially blocked by both paxilline and TRAM-34, specific blockers of large-conductance "maxi-K" (BK) and intermediate K (IK), respectively, and almost abolished by incubation with both antagonists. Apical TRAM-34-sensitive K(+) currents were also observed in BK-null parotid acini. In contrast, when the [Ca(2+)] was increased at the basal or lateral PM, no increase in either K(+) or Cl(-) currents was evoked. These data provide strong evidence that K and Cl channels are similarly distributed in the apical PM. Furthermore, both IK and BK channels are present in this domain, and the density of these channels appears higher in the apical versus basolateral PM. Collectively, this study provides support for a model in which fluid secretion is optimized after expression of K channels specifically in the apical PM.     

Basolateral potassium channels of rabbit colon epithelium: role in sodium absorption and chloride secretion

In order to assess the role of different classes of K(+) channels in recirculation of K(+) across the basolateral membrane of rabbit distal colon epithelium, the effects of various K(+) channel inhibitors were tested on the activity of single K(+) channels from the basolateral membrane, on macroscopic basolateral K(+) conductance, and on the rate of Na(+) absorption and Cl(-) secretion. In single-channel measurements using the lipid bilayer reconstitution system, high-conductance (236 pS), Ca(2+)-activated K(+) (BK(Ca)) channels were most frequently detected; the second most abundant channel was a low-conductance K(+) channel (31 pS) that exhibited channel rundown. In addition to Ba(2+) and charybdotoxin (ChTX), the BK(Ca) channels were inhibited by quinidine, verapamil and tetraethylammonium (TEA), the latter only when present on the side of the channel from which K(+) flow originates. Macroscopic basolateral K(+) conductance, determined in amphotericin-permeabilised epithelia, was also markedly reduced by quinidine and verapamil, TEA inhibited only from the lumen side, and serosal ChTX was without effect. The chromanol 293B and the sulphonylurea tolbutamide did not affect BK(Ca) channels and had no or only a small inhibitory effect on macroscopic basolateral K(+) conductance. Transepithelial Na(+) absorption was partly inhibited by Ba(2+), quinidine and verapamil, suggesting that BK(Ca) channels are involved in basolateral recirculation of K(+) during Na(+) absorption in rabbit colon. The BK(Ca) channel inhibitors TEA and ChTX did not reduce Na(+) absorption, probably because TEA does not enter intact cells and ChTX is 'knocked off' its extracellular binding site by K(+) outflow from the cell interior. Transepithelial Cl(-) secretion was inhibited completely by Ba(2+) and 293B, partly by quinidine but not by the other K(+) channel blockers, indicating that the small (<3 pS) K(V)LQT1 channels are responsible for basolateral K(+) exit during Cl(-) secretion. Hence different types of K(+) channels mediate basolateral K(+) exit during transepithelial Na(+) and Cl(-) transport.     

KCl reabsorption by the lower malpighian tubule of rhodnius prolixus: inhibition by Cl(-) channel blockers and acetazolamide

Iono- and osmoregulation by the blood-feeding hemipteran Rhodnius prolixus involves co-ordinated actions of the upper and lower Malpighian tubules. The upper tubule secretes ions (Na(+), K(+), Cl(-)) and water, whereas the lower tubule reabsorbs K(+) and Cl(-) but not water. The extent of KCl reabsorption by the lower tubule in vitro was monitored by ion-selective microelectrode measurement of Cl(-) and/or K(+) concentration in droplets of fluid secreted by Malpighian tubules isolated under oil. An earlier study proposed that K(+) reabsorption involves an omeprazole-sensitive apical K(+)/H(+) ATPase and Ba(2+)-sensitive basolateral K(+) channels. This paper examines the effects acetazolamide and of compounds that inhibit chloride channels, Cl(-)/HCO(3)(-) exchangers and Na(+)/K(+)/2Cl(-) or K(+)/Cl(-) co-transporters. The results suggest that Cl(-) reabsorption is inhibited by acetazolamide and by Cl(-) channel blockers, including diphenylamine-2-carboxylate(DPC) and 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), but not by compounds that block Na(+)/K(+)/Cl(-) and K(+)/Cl(-) co-transporters. Measurements of transepithelial potential and basolateral membrane potential during changes in bathing saline chloride concentration indicate the presence of DPC- and NPPB-sensitive chloride channels in the basolateral membrane. A working hypothesis of ion movements during KCl reabsorption proposes that Cl(-) moves from lumen to cell through a stilbene-insensitive Cl(-)/HCO(3)(-) exchanger and then exits the cell through basolateral Cl(-) channels.     

Electrophysiology of flounder intestinal mucosa. I. Conductance properties of the cellular and paracellular pathways

We evaluated the conductances for ion flow across the cellular and paracellular pathways of flounder intestine using microelectrode techniques and ion-replacement studies. Apical membrane conductance properties are dominated by the presence of Ba-sensitive K channels. An elevated mucosal solution K concentration, [K]m, depolarized the apical membrane potential (psi a) and, at [K]m less than 40 mM, the K dependence of psi a was abolished by 1-2 mM mucosal Ba. The basolateral membrane displayed Cl conductance behavior, as evidenced by depolarization of the basolateral membrane potential (psi b) with reduced serosal Cl concentrations, [Cl]s. psi b was unaffected by changes in [K]s or [Na]s. From the effect of mucosal Ba on transepithelial K selectivity, we estimated that paracellular conductance (Gp) normally accounts for 96% of transepithelial conductance (Gt). The high Gp attenuates the contribution of the cellular pathway to psi t while permitting the apical K and basolateral Cl conductances to influence the electrical potential differences across both membranes. Thus, psi a and psi b (approximately 60 mV, inside negative) lie between the equilibrium potentials for K (76 mV) and Cl (40 mV), thereby establishing driving forces for K secretion across the apical membrane and Cl absorption across the basolateral membrane. Equivalent circuit analysis suggests that apical conductance (Ga approximately equal to 5 mS/cm2) is sufficient to account for the observed rate of K secretion, but that basolateral conductance (Gb approximately equal to 1.5 mS/cm2) would account for only 50% of net Cl absorption. This, together with our failure to detect a basolateral K conductance, suggests that Cl absorption across this barrier involves KCl co-transport.     

Basolateral Ca2+-dependent K+-channels play a key role in Cl- secretion induced by taurodeoxycholate from colon mucosa

The diarrhea associated with malabsorption of bile salts such as the secondary hydrophobic taurodeoxycholate (TDC) may be partly explained by the TDC-induced increase in colon Cl(-) secretion. We, therefore, investigated the effects of TDC (0.5-8 mM) on electrical parameters and electrolyte transport of rat proximal colon mucosa mounted in Ussing chambers. Colonic secretion, measured as short circuit current (I(SC)), progressively increased on mucosal incubation with TDC ranging 0.5-2 mM; up to TDC 2 mM, a spontaneous recovery toward control values with no changes in epithelial resistance (Rt), and lactate dehydrogenase (LDH) release was observed. In contrast, for TDC > 2 mM, I(SC) increased further and the effect was progressive and associated with a significant decrease in the Rt and increased LDH release, implying a cytolytic effect. Mucosal preincubation with the Cl(-) channel inhibitor 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), fully prevented the precytolytic effect of TDC on I(SC). Serosal preincubation with furosemide, a Na(+)/K(+)/2Cl(-) cotransporter inhibitor, significantly reduced TDC-induced increase in I(SC). Inhibition of the basolateral Ca(2+)-dependent K(+) channel-rSK4-with serosal clotrimazole or incubation with mucosal Ca(2+)-free (EGTA) buffer completely prevented precytolytic TDC-induced increase in I(SC). In conclusion, Cl(-) secretion is activated in colon mucosa by TDC low concentrations; while at higher concentrations, a detergent cytotoxic effect intervenes. Activation of the Ca(2+)-dependent basolateral K(+) pathway, through TDC-induced apical Ca(2+) influx, provides the Na(+)/K(+)/2Cl(-) basolateral activation, thereby the driving force for the apical exit of Cl(-) ions. These findings further enhance the knowledge of the pathogenic mechanisms of diarrhea associated with bile salt malabsorption.     

Characteristics of Kcnn4 channels in the apical membranes of an intestinal epithelial cell line

Intermediate-conductance K(+) (Kcnn4) channels in the apical and basolateral membranes of epithelial cells play important roles in agonist-induced fluid secretion in intestine and colon. Basolateral Kcnn4 channels have been well characterized in situ using patch-clamp methods, but the investigation of Kcnn4 channels in apical membranes in situ has been hampered by a layer of mucus that prevents seal formation. In the present study, we used patch-clamp methods to characterize Kcnn4 channels in the apical membrane of IEC-18 cells, a cell line derived from rat small intestine. A monolayer of IEC-18 cells grown on a permeable support is devoid of mucus, and tight junctions enable selective access to the apical membrane. In inside-out patches, Ca(2+)-dependent K(+) channels observed with iberiotoxin (a Kcnma1/large-conductance, Ca(2+)-activated K(+) channel blocker) and apamin (a Kcnn1-3/small-conductance, Ca(2+)-activated K(+) channel blocker) present in the pipette solution exhibited a single-channel conductance of 31 pS with inward rectification. The currents were reversibly blocked by TRAM-34 (a Kcnn4 blocker) with an IC(50) of 8.7 ± 2.0 μM. The channels were not observed when charybdotoxin, a peptide inhibitor of Kcnn4 channels, was added to the pipette solution. TRAM-34 was less potent in inhibiting Kcnn4 channels in patches from apical membranes than in patches from basolateral membranes, which was consistent with a preferential expression of Kcnn4c and Kcnn4b isoforms in apical and basolateral membranes, respectively. The expression of both isoforms in IEC-18 cells was confirmed by RT-PCR and Western blot analyses. This is the first characterization of Kcnn4 channels in the apical membrane of intestinal epithelial cells.     

The small conductance K+ channel, KCNQ1: expression, function, and subunit composition in murine trachea

The gene KCNQ1 encodes a K(+) channel alpha-subunit important for cardiac repolarization, formerly known as K(v)LQT1. In large and small intestine a channel complex consisting of KCNQ1 and the beta-subunit KCNE3 (MiRP2) is known to mediate the cAMP-activated basolateral K(+) current, which is essential for luminal Cl(-) secretion. Northern blot experiments revealed an expression of both subunits in lung tissue. However, previous reports suggested a role of KCNE1 (minK, Isk) but not KCNE3 in airway epithelial cells. Here we give evidence that KCNE1 is not detected in murine tracheal epithelial cells and that Cl(-) secretion by these cells is not reduced by the knock-out of the KCNE1 gene. In contrast we show that a complex consisting of KCNQ1 and KCNE3 probably forms a basolateral K(+) channel in murine tracheal epithelial cells. As described for colonic epithelium, the current through KCNQ1 complexes in murine trachea is specifically inhibited by the chromanol 293B. A 293B-sensitive current was present after stimulation with forskolin and agonists that increase Ca(2+) as well as after administration of the pharmacological K(+) channel activator, 1-EBIO. A 293B-inhibitable current was already present under control conditions and reduced after administration of amiloride indicating a role of this K(+) channel not only for Cl(-) secretion but also for Na(+) reabsorption. We conclude that at least in mice a KCNQ1 channel complex seems to be the dominant basolateral K(+) conductance in tracheal epithelial cells.     

Synthetic chloride channel restores glutathione secretion in cystic fibrosis airway epithelia

Cystic fibrosis (CF), an inherited disease characterized by defective epithelial Cl- transport, damages lungs via chronic inflammation and oxidative stress. Glutathione, a major antioxidant in the epithelial lung lining fluid, is decreased in the apical fluid of CF airway epithelia due to reduced glutathione efflux (Gao L, Kim KJ, Yankaskas JR, and Forman HJ. Am J Physiol Lung Cell Mol Physiol 277: L113-L118, 1999). The present study examined the question of whether restoration of chloride transport would also restore glutathione secretion. We found that a Cl- channel-forming peptide (N-K4-M2GlyR) and a K+ channel activator (chlorzoxazone) increased Cl- secretion, measured as bumetanide-sensitive short-circuit current, and glutathione efflux, measured by high-performance liquid chromatography, in a human CF airway epithelial cell line (CFT1). Addition of the peptide alone increased glutathione secretion (181 +/- 8% of the control value), whereas chlorzoxazone alone did not significantly affect glutathione efflux; however, chlorzoxazone potentiated the effect of the peptide on glutathione release (359 +/- 16% of the control value). These studies demonstrate that glutathione efflux is associated with apical chloride secretion, not with the CF transmembrane conductance regulator per se, and the defect of glutathione efflux in CF can be overcome pharmacologically.     

Saliva secretion and ionic composition of saliva in the cockroach Periplaneta americana after serotonin and dopamine stimulation,and effects of ouabain and bumetamide

Isolated salivary glands of Periplaneta americana were used to measure secretion rates and, by quantitative capillary electrophoresis, Na(+), K(+), and Cl(-) concentrations in saliva collected during dopamine (1 micro M) and serotonin (1 micro M) stimulation in the absence and presence of ouabain (100 micro M) or bumetanide (10 micro M). Dopamine stimulated secretion of a NaCl-rich hyposmotic saliva containing (mM): Na(+) 95 +/- 2; K(+) 38 +/- 1; Cl(-) 145 +/- 3. Saliva collected during serotonin stimulation had a similar composition. Bumetanide decreased secretion rates induced by dopamine and serotonin; secreted saliva had lower Na(+), K(+) and Cl(-) concentrations and osmolarity. Ouabain caused increased secretion rates on a serotonin background. Saliva secreted during dopamine but not serotonin stimulation in the presence of ouabain had lower K(+) and higher Na(+) and Cl(-) concentrations, and was isosmotic. We concluded: The Na(+)-K(+)-2Cl(-) cotransporter is of cardinal importance for electrolyte and fluid secretion. The Na(+)/K(+)-ATPase contributes to apical Na(+) outward transport and Na(+) and K(+) cycling across the basolateral membrane in acinar P-cells. The salivary ducts modify the primary saliva by Na(+) reabsorption and K(+) secretion, whereby Na(+) reabsorption is energized by the basolateral Na(+)/K(+)-ATPase which imports also some of the K(+) needed for apical K(+) extrusion.     

Cellular mechanism for potentiation of Ca2+-mediated Cl- secretion by the flavonoid baicalein in intestinal epithelia

Flavonoids belong to a large group of plant polyphenols that are consumed daily in large amounts. Our previous findings have shown that baicalein, a major flavonoid derived from the medicinal herb Scutellariae radix, induces Cl(-) secretion across rat colonic mucosa. The current study examines the effect of baicalein on Cl(-) secretion in human colonic epithelial (T84) cells and its interaction with Ca(2+)- and cAMP-dependent secretagogues. We have employed a technique that allows concurrent monitoring of short-circuit current (I(SC)) and [Ca(2+)](i) in polarized epithelium. Basolateral application of baicalein induced a concentration-dependent increase in I(SC). The increase in I(SC) was because of Cl(-) secretion and was not accompanied by any discernible increase in [Ca(2+)](i). Baicalein acted synergistically with Ca(2+)- but not cAMP-dependent secretagogues. In the presence of baicalein, the carbachol and histamine induced increases in I(SC) that were markedly potentiated while increases in [Ca(2+)](i) were not significantly enhanced. Baicalein treatment uncoupled Cl(-) secretion from inhibitory effects normally generated by muscarinic activation. Baicalein treatment also resulted in increased cAMP content and activated PKA activity. Nystatin permeabilization studies revealed that baicalein stimulated an apical Cl(-) current but did not activate any basolateral K(+) current. These data suggest that baicalein potentiates Ca(2+)-mediated Cl(-) secretion through a signaling pathway involving cAMP and protein kinase A, most likely through the cystic fibrosis transmembrane conductance regulator in the apical membrane.     

Small conductance potassium channels drive ATP-activated chloride secretion in the oviduct

The molecular mechanisms controlling fluid secretion within the oviduct have yet to be determined. As in other epithelia, both secretory and absorptive pathways are likely to work in tandem to drive appropriate ionic movement to support fluid movement across the oviduct epithelium. This study explored the role of potassium channels in basolateral extracellular ATP (ATP(e))-stimulated ion transport in bovine oviduct epithelium using the Ussing chamber short-circuit current (I(SC)) technique. Basal I(SC) in bovine oviduct epithelium comprises both chloride secretion and sodium absorption and was inhibited by treatment with basolateral K(+) channel inhibitors tetrapentlyammonium chloride (TPeA) or BaCl(2). Similarly, ATP-stimulated chloride secretion was significantly attenuated by pretreatment with BaCl(2,) tetraethylammonium (TEA), tolbutamide, and TPeA. Basolateral K(+) current, isolated using nystatin-perforation technique, was rapidly activated by ATP(e), and pretreatment of monolayers with thapsigargin or TPeA abolished this ATP-stimulated K(+) current. To further investigate the type of K(+) channel involved in the ATP response in the bovine oviduct, a number of specific Ca(2+)-activated K(+) channel inhibitors were tested on the ATP-induced ΔI(SC) in intact monolayers. Charbydotoxin, (high conductance and intermediate conductance inhibitor), or paxilline, (high conductance inhibitor) did not significantly alter the ATP(e) response. However, pretreatment with the small conductance inhibitor apamin resulted in a 60% reduction in the response to ATP(e). The presence of small conductance family member KCNN3 was confirmed by RT-PCR and immunohistochemistry. Measurements of intracellular calcium using Fura-2 spectrofluorescence imaging revealed the ability of ATP(e) to increase intracellular calcium in a phospholipase C-inositol 1,4,5-trisphosphate pathway-sensitive manner. In conclusion, these results provide strong evidence that purinergic activation of a calcium-dependent, apamin-sensitive potassium conductance is essential to promote chloride secretion and thus fluid formation in the oviduct.     

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.