Effect of histamine on electrical properties in canine tracheal epithelium]

To characterize the action of histamine on ion transport across the airway mucosa, we measured the electrical properties of cultured tracheal epithelium from dogs by Ussing's short-circuited technique in vitro. The addition of histamine to the submucosal side increased short-circuit current (Isc), whereas mucosal addition of histamine had no effect. The histamine-induced increase in Isc was dose-dependent with the maximal increase from the baseline value and EC50 being 4.4 +/- 0.5 microA/cm2 and 10(-6) M, respectively. We also tested the effects of pharmacological blocking agents on the histamine-induced Isc increase. The effect of histamine on Isc was partially inhibited by pretreatment of cells with amiloride, furosemide and diphenylamine carboxylate. Furthermore, the effect of histamine was inhibited by pyrilamine, but not by cimetidine. To determine the contributions of several intracellular second messenger systems to the histamine-induced increase in Isc, we studied the change of Isc by pretreatment of cells with indomethacin, mepacrine and H-7. The increase in Isc produced by histamine was inhibited by indomethacin and mepacrine but not H-7. These results suggest that histamine stimulates both Cl secretion and Na absorption and may affect the subsequent movement of water across the airway epithelium through the activation of submucosal H1-receptor probably involving prostaglandin synthesis.     

Endothelin stimulates ciliary beat frequency and chloride secretion in canine cultured tracheal epithelium

Endothelin, an endothelium-derived vasoconstrictor peptide, has recently been reported to exist in airway epithelial cells. To elucidate a possible role of endothelin on epithelial functions, we studied the effects of this peptide on ciliary beat frequency (CBF) and electrical properties of canine cultured tracheal epithelium by a microphoto-oscillation method and Ussing's short-circuited technique, respectively. Endothelin dose-dependently increased CBF, the maximal increase above the baseline value and EC50 being 32.3 +/- 4.0% (P less than 0.001) and 3 nM, respectively. This effect was moderately attenuated by pretreatment of cells with indomethacin and greatly reduced by Ca2(+)-free medium. Addition of endothelin (10(-6) M) to the mucosal bath of Ussing chamber increased short-circuit current from 5.1 +/- 1.2 to 9.4 +/- 1.7 microA/cm2 (P less than 0.05) and potential difference from 2.9 +/- 0.4 to 5.0 +/- 0.9 mV (P less than 0.05), an effect that was inhibited by indomethacin or Ca2(+)-deficiency and was abolished by the Cl transport inhibitor bumetanide or substitution of Cl with iodide in the medium. These results indicate that endothelin stimulates ciliary motility and Cl secretion probably through an increase in cytosolic Ca2+ and partially a prostaglandin synthesis in canine tracheal epithelium, and suggest that this peptide might play a role in modulating airway mucociliary transport functions.     

Epithelial ion transport of human nasal polyp and paranasal sinus mucosa

Nasal cavity and paranasal sinus have various functions. However, little information is available on ion transport in these upper airway epithelia. In the present study, we measured the anion secretion and the anion channel activity to characterize the ion transport in epithelial cells prepared from human paranasal sinus mucosa (PSM) and nasal polyp (NP). To estimate the anion secretion and the anion channel activity, we measured the short-circuit current (Isc) and the transepithelial conductance (Gt) sensitive to NPPB (a Cl(-) channel blocker). The NPPB-sensitive Isc in PSM was larger than that in NP, correlating to the NPPB-sensitive Gt (Cl(-) channel activity). Forskolin stably elevated the NPPB-sensitive Isc associated with an increase in the NPPB-sensitive Gt in PSM and NP. UTP transiently stimulated the Isc associated with an elevation of Gt in PSM and NP. The stimulatory action of UTP on Isc and Gt was diminished by application of NPPB but not benzamil in PSM and NP, suggesting that UTP induced the NPPB-sensitive Isc (Cl(-) secretion) and Gt (Cl(-) channel activity). These observations suggest that in human PSM and NP, cAMP stably stimulates anion secretion by activating the Cl(-) (anion) channels, and that UTP just transiently elevates anion secretion via activation of some Cl(-) (anion) channels.     

Effects of platelet-activating factor on bioelectric properties of cultured tracheal and bronchial epithelia

To elucidate the effect of platelet-activating factor (PAF) on ion transport function of airway epithelial cells, we studied bioelectric properties of cultured tracheal and bronchial epithelia from dogs under short-circuit conditions in vitro. Addition of PAF (10(-5) mol/L) to mucosal solution of Ussing chamber increased short-circuit current of tracheal epithelium from 3.3 +/- 0.7 to 8.5 +/- 1.2 microA/cm2 (p less than 0.001). This effect was dose dependent, and there was a corresponding increase in transepithelial potential difference. In contrast, PAF was without effect when it was added to the submucosal side. Electrical properties of bronchial epithelium remained unchanged by PAF. The PAF-induced increase in short-circuit current was not affected by amiloride but abolished by diphenylamine-2-carboxylate, bumetanide, or Cl-free medium. The effects of PAF were not altered by AA-861 or U-60257, but attenuated by indomethacin and piroxicam, and dose-dependently blocked by CV 6209 and WEB 2086. Mucosal, but not submucosal, addition of PAF increased the rate of prostaglandin release from tracheal epithelium. These results suggest that PAF selectively stimulates Cl secretion across tracheal epithelium, probably through activation of its specific receptors and the subsequent production of prostaglandins.     

HCO3 – potentiates the cAMP-dependent secretory response of the human distal colon through a DIDS-sensitive pathway

We used the Ussing short-circuit technique to investigate the role of HCO3- in the adenosine 3',5'-cyclic monophosphate (cAMP)-dependent secretory response of the human distal colon. In HCO3(-)-free 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid (HEPES)-Ringer's, forskolin (10 mumol l-1 mucosal and serosal) evoked a sustained increase in short-circuit current (Isc) (delta Isc = 24 +/- 3 microA cm-2, n = 57). However, this was only 25% of the forskolin-stimulated Isc response in HCO3(-)-Ringer's (delta Isc = 84 +/- 8 microA cm-2, n = 57). The reduced response to forskolin in HCO3(-)-free HEPES-Ringer's was not due to inhibition of the secretory mechanism by HEPES, as replacing HCO3- with a different buffer, N-tris[hydroxymethyl)methyl-2-aminoethanesulphonic acid (TES), had a similar effect and inclusion of HEPES in the HCO3(-)-Ringer's did not reduce the secretory response. Similarly, it was not due to an indirect modulation of electrogenic Cl- secretion, as the forskolin-stimulated bumetanide-sensitive Isc was comparable in the two Ringer's. Rather it was due to the activation of a HCO3(-)-dependent Isc which was inhibited by serosal 4,4'-diisothiocyano-stilbene-2,2'-disulphonate (DIDS). This DIDS-sensitive Isc was not inhibited by acetazolamide, but it was inhibited by the replacement of bathing solution Cl- with gluconate, suggesting a role for a Na(+)-dependent Cl-/HCO3- exchanger in the cAMP-dependent secretory response of the human distal colon.     

Alternation of ion transport of airway epithelium by eicosanoids]

BACKGROUND: Airway surface liquid is thought to be regulated by the ion transport processes across the airway epithelium in a vectorial manner. Short circuit current (Isc) measured by Ussing chamber is an indication of electric permeability because of mutually related transportation of amount of ion between apical and basolateral membrane. METHODS: Guinea pig trachea was mounted on Ussing chamber and short-circuited current was determined continuously. After basolateral application of eicosanoids such as PGD2 and LTC4/LTD4/LTE4, Ramatroban, CRTH2 receptor antagonist and Pranlucast, LT receptor antagonist was added from basolateral side. A typical Cl-channel blocker, NPPB, was added from apical side. RESULTS: Isc were increased after basolateral application of eicosanoids. These effects were partly inhibited by each antagonist. Cl- channel blocker suppressed effects of each eicosanoids. CONCLUSION: Eicosanoids alter the ion transport mechanism via chloride channels on the airway epithelium that can be blocked by those antagonists.     

Immunoregulation of endometrial and jejunal epithelia sensitized by infection

The hypothesis was tested that the uterus of the rat orally infected with the parasite Trichinella spiralis becomes hypersensitized and that subsequent antigenic challenge affects functions in the endometrial epithelium. Results of experiments comparing the immunological responsiveness of isolated rat uterus with that of the jejunum supports our hypothesis. Antigenic challenge of uterus mounted in Ussing-type chambers causes an elevation in transuterine short circuit current (Isc) of 6.4 +/- 0.8 microA/cm2. The transduction of the antigenic signal to elicit the electrophysiological response involves 5-hydroxytryptamine (5-HT) working through a nerve-independent pathway. The antigen-stimulated rise in Isc peaks approximately 3 min after challenge. The uterine response is blocked by diisothiocyanostilbene-2,2'-disulfonic acid, an inhibitor of bicarbonate-chloride exchange. The antigen-evoked change in jejunal Isc is biphasic, peaking at 1.5 and approximately 4.0 min after challenge, and is about 10-fold greater in magnitude than the Isc in the uterus. The transductive pathway in the jejunum involves 5-HT, histamine and prostaglandin acting partly through intrinsic nerves. The jejunal response to antigen is inhibited by diphenylamine-2-carboxylate, a chloride channel blocker. Changes in net ion transport which are primed by infection and evoked by antigen are apparently triggered by local anaphylaxis in both the uterus and jejunum.     

Effect of neutral endopeptidase inhibition on substance-P-induced increase in short-circuit current of canine cultured tracheal epithelium.

We studied the effect of substance P (SP) on the electric properties of cultured canine tracheal epithelium and its possible modulation by neutral endopeptidase (NEP) by Ussing's short-circuited technique in vitro. Addition of SP (5 x 10(-6) M) to the mucosal side increased short-circuit current (SCC) from 5.1 +/- 0.9 to 10.3 +/- 2.2 microA/cm2 (mean +/- SE; p less than 0.01), which was accompanied by increases in transepithelial potential difference and conductance. The effect of the mucosal SP on SCC was dose-dependent, with the maximal increase from the baseline value being 5.8 +/- 1.0 microA/cm2 observed at 5 x 10(-5) M. The NEP inhibitor phosphoramidon (10(-5) M) did not affect these responses. On the other hand, SCC was not altered by the addition of SP to the submucosal side. However, it was increased dose-dependently in the presence of phosphoramidon (10(-5) M) but not in the presence of captopril, bestatin or leupeptin. This stimulatory effect of submucosal SP was abolished by furosemide, diphenylamine-2-carboxylate and Cl-free medium, but not by amiloride. These results suggest that SP may selectively stimulate Cl secretion across the airway epithelium and that this effect may be modulated by submucosal NEP.     

Electrophysiological properties of the airway: epithelium in the murine, ovalbumin model of allergic airway disease

The electrophysiological properties of cultured tracheal cells (CTCs) were examined in a murine (C57BL/6J), ovalbumin (OVA)-induced model of allergic airway disease (AAD) at early (3-day OVA-aerosol) and peak (10-day OVA-aerosol) periods of inflammation. Transepithelial potential difference, short-circuit current (Isc), and resistance (RT) were lower in CTCs from 10-day OVA-aerosol animals compared to CTCs from na?ve mice. In cells cultured for 5 weeks, RT was greater in naive CTCs than in 10-day OVA-aerosol CTCs at all times (P < 0.01). The Isc response to mucosal amiloride (10(-4) mol/L) was increased in CTCs from 10-day OVA-aerosol mice compared to na?ve mice (6.0 +/- 0.37 microA/cm2 versus 1.8 +/- 0.56 microA/cm2; P < 0.001) with intermediate values for CTCs from 3-day OVA-aerosol mice. The cAMP-induced increase in Isc was blunted in 10-day OVA-aerosol animals compared to CTCs from na?ve mice (9 +/- 12% versus 39 +/- 7%; P < 0.01) with intermediate values for CTCs from 3-day OVA-aerosol mice. There was no difference in mannitol flux in na?ve compared to 10-day OVA-aerosol CTCs. Similar results were found using intact tracheas mounted in a perfusion chamber. These data demonstrate changes in airway epithelial cell function in the OVA-induced model of AAD that may contribute to the pathogenesis of airway inflammation.     

Deoxycholic acid (DOC) affects the transport properties of distal colon

Secondary bile acids can induce diarrhea. In the present study we examined the effects of deoxycholic acid (DOC) on equivalent short-circuit current (Isc) in rabbit colon and the cellular mechanisms involved in DOC action (rabbit and rat). Luminal DOC inhibited amiloride-sensitive Na+ absorption. In the presence of amiloride luminal DOC had a concentration dependent effect on Isc. Low concentrations (1-10 micromol/l) induced a lumen-positive current (51+/-3 microA/cm2, 10 micromol/l, n=7) which was inhibited by luminal Ba2+ suggesting the activation of a luminal K+ conductance. Higher luminal concentrations induced a lumen-negative current (-76+/-9 microA/cm2, 100 micromol/l, n=11). Basolateral application of DOC, also in the presence of amiloride, only induced lumen-negative Isc, (-58+/-10 microA/cm2, 100 micromol/l, n=6, EC50= 3 micromol/l). This current could be abolished completely by the K+ channel blocker 293B, a selective inhibitor of cAMP-dependent Cl- secretion. This action of DOC on Isc was additive to the effect of carbachol (CCH) but not additive to that of cAMP. In intact rat colon mucosa pre-treated with DOC a significant increase in cAMP production was observed. Fura-2 measurements of cytosolic Ca2+ activity ([Ca2+]i) in isolated colonic crypts (rabbit and rat) showed that 100 micromol/l DOC induced a weak [Ca2+]i increase. Whole-cell measurements of membrane voltage in isolated rat colonic crypts revealed a hyperpolarization by DOC (4.9+/-0.8 mV, 100 micromol/l, n=8) but a depolarization by prostaglandin E2 (PGE2, via cAMP) (24+/-7 mV, n=8). The present data show that DOC acts at more than one target in the colon: in the intact mucosa it activates luminal K+ channels and Cl- secretion and this is paralleled by an increase in cAMP production. In isolated crypts DOC probably activates a Ca(2+)-regulated K+ conductance but has no effect on cAMP. Hence DOC probably activates ion channels or channel-regulating factors in colonocytes and acts on non-epithelial cells to activate Cl- secretion indirectly.     

Differential regulations between adenosine triphosphate (ATP)- and uridine triphosphate-induced Cl(-) secretion in bovine tracheal epithelium. Direct stimulation of P1-like receptor by ATP

Adenosine 5'-triphosphate (ATP) stimulates airway epithelial Cl(-) secretion in a complicated manner. We examined the difference between ATP- and uridine 5'-triphosphate (UTP)-induced responses of short-circuit current (Isc) in bovine tracheal epithelium treated with amiloride. Each nucleotide caused an increase in Isc composed of the first and second peaks, where the second peak induced by ATP was higher compared with UTP. The ATP-induced second peak was inhibited by the protein kinase (PK) A inhibitor H89, saturation of P1 receptor with adenosine, and the P1 receptor antagonist 8-p-sulfophenyltheophylline, but not by the Ca(2+) chelator ethyleneglycol-bis-(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid plus the endoplasmic reticulum Ca(2+)-pump inhibitor thapsigargin, the adenosine breakdown enzyme adenosine deaminase, the ectonucleotidase inhibitor alpha,beta-methyleneadenosine 5'-diphosphate, or saturation of P2Y2 receptor with UTP. Thus, the response is associated with PKA-dependent pathway via P1-like receptor but not with Ca(2+)-dependent pathway via P2Y2 receptor, and ATP degradation products do not contribute to this response. Further, stimulation of cells with ATP increased PKA activity. In addition, pretreatment with glybenclamide, an inhibitor of cystic fibrosis transmembrane conductance regulator, reduced the second peak of Isc induced by ATP but was without effect on that induced by UTP. Therefore, ATP stimulates glybenclamide-sensitive Cl(-) secretion, and this action is partly mediated by PKA-dependent pathway via P1-like receptor.     

Dopamine-induced inhibition of Na+-K+-ATPase activity requires integrity of actin cytoskeleton in opossum kidney cells

The present study evaluated the importance of the association between Na+-K+-ATPase and the actin cytoskeleton on dopamine-induced inhibition of Na+-K+-ATPase activity. The approach used measures the transepithelial transport of Na+ in monolayers of opossum kidney (OK) cells, when the Na+ delivered to Na+-K+-ATPase was increased at the saturating level by amphotericin B. The maximal amphotericin B (1.0 microg mL-1) induced increase in short-circuit current (Isc) was prevented by ouabain (100 microM) or removal of apical Na+. Dopamine (1 microM) applied from the apical side significantly decreased (29 +/- 5% reduction) the amphotericin B-induced increase in Isc, this being prevented by the D1-like receptor antagonist SKF 83566 (1 microM) and the protein kinase C (PKC) inhibitor chelerythrine (1 microM). Exposure of OK cells to cytochalasin B (1 microM) or cytochalasin D (1 microM), inhibitors of actin polymerization, from both cell sides reduced by 31 +/- 4% and 36 +/- 3% the amphotericin B-induced increase in Isc and abolished the inhibitory effect of apical dopamine (1 microM), but not that of the PKC activator phorbol-12,13-dibutyrate (PDBu; 100 nM). Colchicine (1 microM) failed to alter the inhibitory effects of dopamine. The relationship between Na+-K+-ATPase and the concentration of extracellular Na+ showed a Michaelis-Menten constant (Km) of 44.1 +/- 13.7 mM and a Vmax of 49.6 +/- 4.8 microA cm-2 in control monolayers. In the presence of apical dopamine (1 microM) or cytochalasin B (1 microM) Vmax values were significantly (P < 0.05) reduced without changes in Km values. These results are the first, obtained in live cells, showing that the PKC-dependent inhibition of Na+-K+-ATPase activity by dopamine requires the integrity of the association between actin cytoskeleton and Na+-K+-ATPase.     

Cellular signaling mechanisms underlying pharmacological action of Bak Foong Pills on gastrointestinal secretion

Bak Foong Pills (BFP, also known as Bai Feng Wan) is an over-the-counter traditional Chinese medicine that has long been used for treating gynecological disorders and improving overall body functions, including gastrointestinal (GI) function. However, the cellular signaling mechanism underlying BFP action, especially on the GI tract, has not been elucidated. In the present study, the human colonic epithelia cell line T(84) was used as a model to investigate the effect of BFP ethanol extract on ion transport in conjunction with the short-circuit current (I(SC)) technique. The results showed that the apical addition of BFP extract produced a concentration-dependent (10-1,000 microg/ml, EC(50) = 120 microg/ml) increase in I(SC). The maximal response was observed at 500 microg/ml with an increase in I(SC) of 24.4 +/- 2.3 microA/cm(2) and apical conductance. The BFP-induced I(SC) was not observed when extracellular Cl(-) was replaced or when treated with Bumetanide (100 microM), an inhibitor of the Na(+)-K(+)-2Cl(-) cotransporter. The BFP-induced I(SC) was insensitive to the Na(+) channel blocker, amiloride, but partially inhibited by the Cl(-) channel blocker, DIDS (100 microM), and completely blocked by DPC (2 mM) or glibenclamide (1 mM) with a significant reduction in the apical conductance. The BFP-induced I(SC) could be mimicked by forskolin (10 microM), but inhibited by a pretreatment of the cells with adenylate cyclase inhibitor, MDL-12330A (10 microM). Pretreatment with EGTA (5 mM) and thapsigargin (10 microM) decreased the BFP-induced I(SC) by 10%. These results demonstrated that BFP ethanol extract exerted a stimulatory effect on gastrointestinal Cl(-) secretion by predominantly activating adenylate cyclase and apical cAMP-dependent Cl(-) channels, with minor contributions from calcium-dependent Cl(-) channels. The effect of BFP may be explored to treat GI disorders such as constipation.     

The transmural potential of the newt ureter: evidence for amiloride-sensitive active sodium transport

Transmural electrical potential difference (PD) of the newt ureter was measured with glass microelectrodes and its nature was studied pharmacologically or by ion replacement experiments. The PD in the presence of standard Ringer solution on both sides averaged -76.0 +/- 1.3 mV, lumen negative. The magnitude of the PD depended on the Na+ concentration of the luminal fluid; the PD increased in a saturable way with increase in the Na+ concentration. Amiloride added to the luminal fluid at 10(-4) M greatly reduced the PD and increased the transmural resistance. The half maximum inhibition was seen at 2.6 x 10(-6) M and the maximum inhibition at 10(-4) M where the PD reduced to 3.8% of control. Amiloride added to the external medium at 10(-4) M had no effect on both the PD and the resistance. Ouabain added to the external medium at 10(-3) M caused a gradual decrease in the PD, which fell to 36% of its initial value after 60 min. The specific transmural resistance (Rt) was estimated by recording spatially decaying electrotonic potentials along the ureter and applying cable analysis. The short-circuit current (Isc) was calculated from the PD and the Rt. The Rt and the Isc averaged 31.7 +/- 7.7 k omega X cm2 and 3.9 +/- 1.5 microA X cm-2, respectively. The results indicate that the epithelial cells of the newt ureter have amiloride-sensitive Na+ channels at the luminal membrane and are transporting Na+ actively, like epithelia of the urinary bladder and the skin of amphibia.     

Mechanism of electrically silent Na and Cl transport across the rumen epithelium of sheep

This study was designed to study the mechanism of electroneutral Na and Cl transport across the isolated rumen epithelium of sheep. Net sodium transport (5.75 +/- 0.35 microequiv cm-2 h-1) was significantly higher than the short-circuit current (0.95 +/- 0.08 microequiv cm-2 h-1). Both, net sodium and net chloride transport were markedly reduced by replacement of chloride, bicarbonate and sodium, respectively, but were not changed by the absence of mucosal potassium. Net sodium and net chloride absorption was significantly decreased by 1.0 mM-amiloride. Mucosal addition of bumetanide, furosemide, hydrochlorothiazide or low concentrations of amiloride (less than 0.1 mM) did not change sodium fluxes. These results provide compelling evidence consistent with the presence of Na-H exchange as the predominant electroneutral mechanism for transepithelial sodium movement. The ion replacement studies and data from literature suggest that the Na-H exchange is working in parallel with a Cl-HCO3 exchange although luminal addition of DIDS (4,4'diisothiocyanatostilbene-2,2'-disulphonate, 1 mM) did not significantly influence Cl transport.     

Arginine vasopressin stimulation of chloride secretion in Madin-Darby canine renal epithelial layers

Arginine vasopressin (AVP) at a concentration of 0.1 microM stimulates an inward short-circuit current (SCC) in voltage-clamped Madin-Darby canine kidney epithelial layers with a mean peak of 3.1 +/- 0.8 microA cm-2 (n = 6). The response to AVP is markedly attenuated when inward SCC is stimulated with forskolin or prostaglandin E1, in Cl(-)-free media and by the Cl(-)-transport inhibitors, bumetanide and 5-nitro-2-(3-phenylpropylamino)-benzoic acid, consistent with an AVP-stimulated basal to apical Cl- secretion. Endogenous release of prostaglandins may contribute to AVP stimulation of Cl- secretion.     

The myeloid leukemia cell line HL-60 produces a factor that induces chloride secretion in cultured epithelial cells.

When activated, polymorphonuclear leukocytes (PMNs) produce a small soluble factor, termed neutrophil-derived secretagogue (NDS), that elicits electrogenic Cl- secretion--the transport event responsible for hydration of mucosal surfaces. Work toward purification of this factor has been hampered by variability in activity of PMN-derived NDS. Using a human-derived intestinal epithelial cell line (T84) that serves as a model for studies of the regulation of electrogenic Cl- secretion, we find that the human promyelocytic leukemia cell line HL-60 secrets a factor with NDS-like activity. Buffer conditioned by HL-60 cells (10(7) cells/ml for 1 h), when applied to T84 monolayers grown on permeable supports and analyzed by routine electrophysiological techniques, elicited a short-circuit current (Isc) of 11.7 +/- 1.02 microA/cm2. This short-circuit current was sensitive to bumetanide, an inhibitor of the basolateral Na-K-2Cl cotransporter, and was dependent on the presence of chloride in the assay buffers. Such data indicate that buffer conditioned by HL-60 cells stimulates electrogenic Cl- secretion. Like NDS, the active factor in HL-60-conditioned buffer has a nominal molecular weight of less than 500 and was increased by activation of cells with phorbol ester. 125I and 86Rb efflux assays confirmed that the secretagogue released by stimulated HL-60 cells, similar to PMN-derived NDS, preferentially stimulates opening of Cl- rather than K+ channels in T84 cells. Lastly, release of NDS-like bioactivity increases when HL-60 cells are differentiated toward granulocytes compared to cells differentiated toward monocytes.     

Methoxsalen stimulates electrogenic Cl- secretion in the mouse jejunum

We used the short-circuit current (I(sc)) and patch-clamp techniques to investigate the effects of methoxsalen (MTX) on the electrogenic Cl- secretion of the mouse jejunum. MTX stimulated a sustained increase in Isc that was dose dependent. Bumetanide inhibited MTX-stimulated Isc in a dose-dependent manner consistent with activation of Cl- secretion. MTX failed to stimulate I(sc) following maximal activation of the cAMP pathway by forskolin, but did increase Isc after a submaximal dose of forskolin. Glibenclamide, a blocker of the cystic fibrosis transmembrane conductance regulator (CFTR), reduced the MTX-stimulated increase of Isc by 59 +/- 6%. The cAMP-dependent K+ channel blocker 293B did not alter the MTX-activated I(sc); however, clotrimazole, an intermediate Ca2(+)-activated K+ channel (IK(Ca)) blocker, reduced the MTX-stimulated I(sc). MTX did not alter Na(+)-glucose cotransport across the mouse jejunum. In cell-attached membrane patches, MTX increased the open probability of the basolateral IK(Ca) channel of isolated crypts. These data suggest that the CFTR and IK(Ca) channels participate in the MTX-activated, sustained Cl- secretory response of the mouse jejunum.     

The use of isolated fish opercular epithelium as a model tissue for studying intrinsic activities of loop diuretics

Isolated opercular epithelia of killifish (Fundulus heteroclitus) were mounted in an Ussing chamber. The epithelia displayed a transepithelial electrical potential difference (PD) of 10.6 +/- 0.3 mV (sea-water side negative) and a short-circuit current (SCC) of 72.1 +/- 2.1 microA cm-2. The electrical resistance was 160 +/- 3 omega cm2 (mean +/- SE, n = 269). The unidirectional flux of 36Cl from blood side to sea-water side compared well with the SCC. No net flux of 22Na or 24Na across the epithelium was observed. Raising of cyclic AMP levels by theophylline, 3-isobutyl-I-methyl-xanthine, isoprenaline and forskolin, increased SCC and PD. Prostaglandins PGE2 and to some extent PGF2 alpha inhibited SCC and PD. Inhibition of Na+-K+-ATPase by ouabain and orthovanadate reduced SCC and PD. Pretreatment of the epithelium with the stilbene disulphonic acid (DIDS) did not prevent the action of orthovanadate. Different types of diuretics were tested, but only the loop diuretics bumetanide, piretanide, and furosemide, rapidly and strongly inhibited PD and SCC and unidirectional 36Cl serosal to mucosal flux. Dose-response curves for these agents were parallel and EC50 values for effects on SCC were 40, 52, and 295 microM, respectively. The same relative activities of these diuretics have been seen in the renal thick ascending limb of Henle's loop (TALH). It is concluded that the killifish opercular skin responds to hormonal stimuli and various pharmacological agents in a manner similar to that of mammalian renal TALH. It should therefore be a useful model for studies of the modes of action and the structure-activity relationships of diuretics which act by inhibition of chloride transport or Na+-K+-ATPase activity.     

Effects of endothelin-1 on epithelial ion transport in human airways

Endothelin-1 (ET-1) exerts many biological effects in airways, including bronchoconstriction, airway mucus secretion, cell proliferation, and inflammation. We investigated the effect of ET-1 on Na absorption and Cl secretion in human bronchial epithelial cells. Addition of 10(-7) M ET-1 had no effect on the inhibition of the short circuit current (Isc) induced by amiloride, a Na channel blocker. Addition of 10(-7) M ET-1 to the apical bath in the presence of amiloride increased Isc in cultured human bronchial epithelial cells studied in Ussing chambers. No effect was observed when ET-1 was added to basolateral bath, indicating that the involved ET-1 receptors are likely present only in the apical membrane of the cells. Use of Cl-free solutions and bumetanide reduced the ET-1-induced increases in Isc, indicating that ET-1 stimulates Cl secretion. The ET-1-induced increase in Isc was prevented by exposure to the ETB receptor antagonist BQ-788 but not to the ETA receptor antagonist BQ-123. ET-1 did not raise intracellular Ca levels, but increased the intracellular concentration of cAMP. These findings indicate that ET-1 is a Cl secretagogue in human airways and acts presumably through apically located ETB receptors and activation of the cAMP pathway.