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.     

Effect of ion transport inhibitors and methacholine on short-circuit current of isolated guinea pig nasal epithelium

To clarify the ion/water secretion mechanism in the nasal epithelial cells, the Ussing chamber method was applied to the nasal mucosa isolated from guinea pigs. The preparation, which contained surface epithelial cells, showed a small but consistent potential difference between mucosal and submucosal sides (mucosal surface negative to submucosa). The short-circuit current (Isc) across the epithelial layer was measured, and the effects of Na+ and/or Cl- transport inhibitors and methacholine (MCh) on Isc were analyzed. The basal Isc was almost totally suppressed by the combined application of amiloride (Na+ transport inhibitor) and low-Cl- Krebs Ringer (KR) solution or solutions containing Cl- transport inhibitors (furosemide or DPC). The application of MCh elicited triphasic Isc responses, i.e., initial transient increase (phase 1) followed by a small decrease (phase 2) and further sustained increase (phase 3) in Isc. A possible ionic mechanism underlying phase 1 and 3 responses was analyzed. The Phase 1 response was greatly reduced by low-Cl- KR solution or furosemide but not influenced by amiloride. The Phase 3 response was augmented by amiloride and suppressed by low-Cl- KR solution, furosemide or DPC. These findings indicated that the basal Isc was associated with Cl- secretion and/or Na+ absorption across epithelial cells under short-circuit condition and that MCh increased Isc probably via enhancing Cl- secretion in the nasal surface epithelial cell.     

Effects of Ca2+ on ileal transport and electrically induced secretion

[Ca2+] affects nerves and target cells in stimulus-secretion coupling. In flux-chamber studies of full-thickness rabbit ileum, we determined the effects of 1) ethylene glycol bis-(beta-aminoethylether)-N,N'-tetraacetic acid (EGTA) 1.25 mM, 2) verapamil 0.1 mM and nifedipine 0.1 mM, and 3) trifluoperazine 0.1 mM on ion transport and its response to electrical field stimulation (EFS). EGTA increased JClm leads to s, JNam leads to s, Cl absorption and conductivity (G), and reduced Isc. In the absence of EGTA, EFS increased transmural PD and Isc and caused secretion of Na and Cl. EGTA prevented the responses to EFS, but the Isc responses to aminophylline and to glucose were normal. Verapamil reduced the response of Isc and Cl transport to EFS. Nifedipine reduced Isc but not the Isc response to EFS. Trifluoperazine reduced Isc and almost eliminated the Isc response to EFS. EFS did not increase the tissue concentration of cAMP. We conclude: 1) low extracellular [Ca2+] enhances net Cl absorption; 2) extracellular Ca2+ is required for the response of ion transport to EFS; 3) cAMP does not mediate Isc response to EFS; and 4) Isc response to EFS is blocked by trifluoperazine. The findings suggest that EFS stimulates secretion by increasing Ca entry into the epithelial cells, either directly or 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.     

Prostaglandin D2 increases Cl secretion across canine tracheal epithelium through cyclo-oxygenase stimulation and cAMP production.

Prostaglandin (PG) D2 is one cyclo-oxygenase product of arachidonic acid metabolites that may play a role in the pathogenesis of asthma. To determine the effect of PGD2 on ion transport by airway epithelium and its mechanism of action, we measured bioelectric properties of canine cultured tracheal epithelium under short-circuit conditions in vitro. PGD2 (10(-7) M) increased short-circuit current (Isc) from 5.5 +/- 1.2 to 14.1 +/- 2.9 microA cm-2 (means +/- SE, P less than 0.01) when added to the mucosal solution, and to 22.2 +/- 3.8 microA cm-2 (P less than 0.001) when added to the submucosal solution, an effect that was accompanied by the corresponding increases in transepithelial potential difference and conductance. These effects were dose-dependent. The PGD2-induced increase in Isc was not altered by preincubation of cells with autonomic antagonists (phentolamine, propranolol, atropine), the lipoxygenase inhibitor AA-861, the protein kinase C inhibitor H-7, or the Na channel blocker amiloride, but it was inhibited by each of indomethacin, piroxicam, the Cl channel blocker diphenylamine-2-carboxylate, the Cl transport inhibitor furosemide, and Cl-free medium. Intracellular adenosine 3',5'-cyclic monophosphate (cAMP) levels were dose-dependently increased by PGD2. These results suggest that PGD2 may selectively stimulate airway epithelial Cl secretion via cyclo-oxygenase- and cAMP-dependent pathway.     

Effects of methylprednisolone on electrolyte transport by in vitro rat ileum

Administration of the glucocorticoid methylprednisolone (MP) (30 mg/kg body wt for 3 days) to rats increased intestinal mucosal guanylate cyclase and Na-K-ATPase activities, short-circuit current (Isc), electrical potential difference (PD), net Na absorption, and net Cl secretion and reversed HCO3 transport from secretion to absorption. In the MP-treated animals, removal of HCO3 from both the mucosal and serosal bathing solutions increased Cl secretion but did not alter the Isc, PD, and net Na flux. Removal of Cl abolished the MP-induced increase in Isc but did not affect the MP-induced changes in net Na and HCO3 fluxes. At 6 h, after a single dose of MP, stimulation of guanylate cyclase activity was already maximal, whereas Na-K-ATPase activity was not detectably altered. The changes in intestinal transport properties present 6 h after MP treatment and associated with the increased guanylate cyclase activity were an increase in Isc and PD and a reversal of net Cl absorption to net secretion. These results suggest that an initial response to MP administration is a persistent increase in intestinal guanylate cyclase activity that mediates an electrogenic Cl secretory process, then is followed by a superimposed effect of increased Na-K-ATPase activity that mediates an increase in net Na absorption.     

Ion and liquid transport across the bronchiolar epithelium

The proper homeostasis of the airway surface liquid (ASL) depends on transepithelial ion and fluid transport and is critically important for lung defence, and more specifically for mucociliary transport. In cystic fibrosis (CF), abnormal ion and fluid transport lead to depleted ASL volume resulting in mucus plugs and recurrent lung infections. Like bronchi, human bronchioles exhibit amiloride-sensitive Na(+) absorption and cyclic-AMP and Ca(2+)-activated Cl(-) secretion. However, cyclic-AMP-stimulated Cl(-) and fluid secretion appears to be quantitatively more important in bronchioles than in bronchi. In CF bronchioles, like in CF bronchi, the ASL height is reduced because of an abnormally persistent Na(+) absorption, combined with a lacking CFTR-dependent Cl(-) secretion. The precocity and severity of the bronchiolar disease in CF could be attributed in part to the more important role of CFTR-dependent Cl(-) secretion and fluid secretion, and the lack of compensatory ATP-driven Cl(-) secretion and fluid secretion, in bronchioles compared to bronchi.     

Volume regulation by Necturus gallbladder: apical Na+-H+ and Cl(-)-HCO-3 exchange

Necturus gallbladder epithelial cells exhibited volume regulatory swelling when exposed to a hypertonic mucosal bathing solution. The initial, osmotically induced shrinkage was followed by a rapid increase in cell volume back to the control value despite continuing hypertonicity of the mucosal perfusate. This volume regulatory increase occurred by osmotic water flow accompanying the transient cellular uptake of NaCl from the mucosal bathing solution. Volume regulatory increase required Na+ and Cl- in the mucosal bath; it was inhibited by amiloride or 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid but not by bumetanide or ouabain. The K1/2 for Na+ was 2.8 mM, the K1/2 for Cl- was 1.9 mM, and maximum velocity of fluid flow into the cell for both ions was greater than 10 x 10(-6) cm/s. Both volume regulatory increase and transepithelial fluid absorption involve NaCl flux across the apical membrane into the cells, but the nature of the NaCl fluxes differ in the two processes. During volume regulatory increase NaCl enters the cells by parallel Na+-H+ and Cl(-)-HCO-3 exchanges, whereas during transepithelial fluid absorption NaCl enters the cell by the coupled flux of NaCl.     

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.     

Transport characteristics of isolated newborn rabbit ileum

Segments of ileum from newborn rabbits were mounted as flat sheets in Lucite chambers and transmural fluxes of Na, Cl, and alanine were measured in the absence of electrochemical potential gradients. In the presence of 140 mM Na, the ileum of the newborn exhibited a serosa-positive electrical PD and a corresponding short-circuit current (Isc) which was markedly enhanced by the addition of D-glucose of L-alanine to the mucosal solution. Alanine-induced increments in Isc were a saturable function of the mucosal alanine concentration. In the presence of Na, alanine was actively absorbed, and the net alanine flux was a saturable function of alanine concentration. When the Na in the bathing solutions was completely replaced by choline, Isc declined to near zero, and the response of Isc to mucosal alanine was abolished. In addition, active amino acid transport was abolished in the absence of Na. In the absence of alanine, the isolated ileum of the newborn actively absorbed Na and Cl, and the algebraic sum of the net movements of these ions accounted for Isc. In the presence of alanine, active Cl transport was abolished and Isc was equal to the net Na absorption. These results indicate that the ileum of the newborn rabbit is similar to that of the adult in its ability to actively absorb Na, Cl, and alanine, but differs from the ileum of the adult by having a greater passive permeability to ions and amino acid, and differs particularly with regard to the effect of mucosal alanine on transmural ion transport.     

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.     

Ion transport across Xenopus alveolar epithelium is regulated by extracellular ATP, UTP and adenosine

Native alveolar epithelium from Xenopus lung was used for electrophysiological Ussing chamber experiments to investigate ion transport regulation. The tissue exhibits a considerable absorption of Na(+) ions and this transepithelial transport is largely up-regulated after treatment of donor animals with ACTH. Extracellular ATP, UTP and adenosine were tested for their regulating effects and all three increased I(sc), which was mainly due to a stimulation of amiloride sensitive Na(+) transport (increase of I(ami) 32% for ATP, 21% for UTP, 25% for adenosine). Solely the effect of UTP was completely abolished in the presence of amiloride. In contrast, the effects of ATP or adenosine disappeared under Cl(-)-free conditions. ATP and UTP proved to have additive effects and pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), an antagonist of purinergic receptors, inhibited selectively the effect of UTP on I(sc). Further, I(sc) was increased by the P2X selective agonist beta,gamma-meATP. We were able to demonstrate, that extracellular purines and pyrimidines play a possible role as auto/paracrine messengers for alveolar ion transport regulation in Xenopus lung.     

Perceptual characteristics of the amiloride-suppressed sodium chloride taste response in the rat

The contribution of amiloride-sensitive membrane components to the perception of NaCl taste was assessed by using a conditioned taste aversion procedure. Eight independent groups of adult rats were conditioned to avoid either 0.1M NaCl, 0.5M NaCl; 0.1M NH4Cl, or 1.0M sucrose while their tongues were exposed either to water or to the sodium transport blocker amiloride hydrochloride. In contrast to rats exposed to water during conditioning, rats exposed to amiloride were unable to acquire a conditioned taste aversion to 0.1M NaCl. Differences in the acquisition of taste aversions between the amiloride- and nonamiloride-treated groups were not apparent when the conditioned stimulus (CS) was 0.5M NaCl, 0.1M NH4Cl, or 1.0M sucrose. Although the magnitude of the 0.5M NaCl aversion was similar between amiloride- and non-amiloride-treated rats, the perceptual characteristics of the CS differed between groups. Analyses of stimulus generalization gradients revealed that amiloride-treated rats generally avoided all monochloride salts after conditioning to 0.5M NaCl but not nonsodium salts or nonsalt stimuli. In contrast, rats not treated with amiloride only generalized the 0.5M NaCl aversion to sodium salts. No differences in generalization gradients occurred between groups when the CS was 0.1M NH4Cl or 1.0M sucrose. These findings suggest that the "salty" taste of NaCl is primarily related to the amiloride-sensitive portion of the functional taste response in rats. Conversely, the portion of the NaCl response insensitive to amiloride appears to have "sour-salty" perceptual characteristics and does not appear to be perceived as being salty.     

Ion transport by amphibian antrum in vitro. I. General characteristics.

Both Necturus and bullfrog antrum show stable PD, resistance, and short-circuit current (Isc) when mounted in an Ussing chamber. Measurements of Na+ and Cl minus flux showed that both ions are actively transported across Necturus antrum, Na+ from secretory to nutrient, Cl minus from nutrient to secretory (both net fluxes being similar to 0.30 mueq cm minus 2 h minus 1). Only the Na+ transport contributed to the Isc and PD as evidenced by a) Na+ removal, b) the effects of amiloride on the secretory surface, c) the effects of ouabain on the nutrient side. Microelectrode experiments confirm the Na+ conductance of the secretory cell membrance, a HCO3 minus conductance of both cell membranes, and a KCl conductance across the nutrient cell membrane. In addition, antrum apparently secretes alkali (similar to 0.35 mueq cm minus 2 h minus 1), which secretion is sensitive to metabolic inhibitors and Diamox. Nutrientside HCO3 minus increased the rate of alkaline secretion and a transmucosal HCO3 minus gradient could contribute to ISC and PD. A model is proposed to account for the electrical properties of the tissue.     

Monocyte/macrophage activation by normal bacteria and bacterial products: implications for altered epithelial function in Crohn's disease

Intestinal immune cells are less reactive than those in the peripheral blood; however, such cells from patients with Crohn's disease may be more responsive to bacterial products. Our study examined if nonpathogenic bacteria or lipopolysaccharide (LPS), can affect epithelial function in the presence of monocytes/macrophages. Lamina propria mononuclear cells (LPMCs) and peripheral blood monocytes (PBMs) were obtained from patients with Crohn's disease and control patients. Filter-grown T84 epithelial monolayers were co-cultured with nonactivated or LPS-activated LPMCs or PBMs for 48 hours. Epithelial secretory [baseline short-circuit current (Isc) and DeltaIsc to forskolin] and barrier (transepithelial electrical resistance) parameters were measured in Ussing chambers. LPS-activated PBMs from both controls and patients with Crohn's disease significantly increased Isc ( approximately 300%) and reduced transepithelial electrical resistance ( approximately 40%). Epithelial function was not altered after co-culture with control LPMCs +/- LPS. However, LPMCs from patients with Crohn's disease spontaneously secreted tumor necrosis factor-alpha, and induced epithelial changes similar to those produced by LPS-activated PBMs. Co-culture with control Escherichia coli and PBMs induced comparable changes in epithelial physiology, which were abrogated by anti-tumor necrosis factor-alpha antibody. We conclude that LPMCs of patients with Crohn's disease are spontaneously activated, possibly by gram-negative luminal bacteria, and can directly cause significant alterations in epithelial ion transport and barrier functions.     

cGMP modulation of ileal ion transport: in vitro effects of Escherichia coli heat-stable enterotoxin

Diarrheagenic strains of Escherichia coli have been shown to produce a heat-stable enterotoxin (ST) that simulates guanylate cyclase, increases short-circuit current (Isc), and inhibits active Cl absorption in the intestine. In rabbit ileum, the ion transport effects are smaller than those produced by cAMP-related agonists. Because ST may be a selective cGMP agonist, we further explored its mode of action in rabbit ileum. ST inhibits net Na and net Cl absorption. ST also inhibits the same fraction of Cl influx across the brush border that theophylline inhibits. At maximal doses, ST and 8-bromo-cGMP (8-Br-cGMP) had nearly equal, nonadditive effects of Isc that were about 66% of that produced by 8-Br-cAMP. ST increased mucosal cGMP concentration 16-fold, whereas epinephrine, an inhibitor of secretion, increased cGMP concentration by only 30%. This is insufficient to alter ion transport because doses of ST that increased cGMP concentration by 100% failed to alter Cl fluxes. Furthermore, epinephrine did not increase cGMP concentration in isolated enterocytes. We conclude that 1) cGMP mediates ST effects on ion transport, and 2) although ST and cAMP-related agonists have the same antiabsorptive effects, ST is less effective in stimulating electrogenic Cl secretion.     

Actions of the Cl- channel blocker NPPB on absorptive and secretory transport processes of Na+ and Cl- in rat descending colon

The effects of the Cl- channel blocker, NPPB (5-nitro-2-(3-phenylpropylamino)-benzoate), on the transport of Na+ and Cl- in the descending colon of the rat were studied in the Ussing chamber. In control tissue, NPPB administered at the mucosal side of the epithelium increased the short-circuit current (Isc) and inhibited the unidirectional mucosa-to-serosa fluxes of Na+ and Cl-. In HCO3- - or Cl- -free media for in the presence of SITS (4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulphonic acid), this increase in Isc caused by mucosal NPPB was not observed. The serosal administration of NPPB was without effect. Mucosal NPPB (10(-4) mol l-1) decreased the forskolin-induced increase in Isc by only about 60%. However, the activation of the serosa-to-mucosa flux of Cl- caused by forskolin was inhibited completely. NPPB decreased the mucosa-to-serosa fluxes of Na+ and Cl- reduced additionally by forskolin. Serosal NPPB decreased Isc and FNasm, but had no effect on FNams or FClmas. In HCO3- -free buffer the increase in Isc induced by forskolin was inhibited completely by NPPB. The inhibition of Cl- secretion by NPPB fits well with the capacity of the drug to block Cl- channels. For the inhibition of neutral NaCl absorption two sites of action are discussed: an interaction with the Cl-/HCO3- exchanger or an interference with the extrusion of Cl- through the basolateral membrane.     

Hypotonicity-induced ATP release is potentiated by intracellular Ca2+ and cyclic AMP in cultured human bronchial cells

We have examined the cultured human bronchial epithelial cells (16HBE) to learn if changes in Cl(-) concentration or osmolality stimulate the cells to release ATP and to determine whether its release is cyclic AMP (cAMP)- and/or Ca(2+)-dependent by using the luciferin-luciferase luminometric assay. In a control solution (290 mosmol kg H(2)O(-1)), the external ATP concentration and the rate of ATP release were 0.52 +/- 0.20 nM and 0.036 +/- 0.034 pmol min(-1), respectively. Upon hypotonicity (205 mosmol kg H(2)O(-1)), they increased to 7.0 +/- 1.3 nM and 3.1 +/- 0.6 pmol min(-1), respectively, at 6 min, then decreased. At the peak, the rate of ATP release is estimated to be 6.2x10(4) ATP molecules s(-1) per cell. An accumulation of the released ATP for the initial 10 min increased significantly (p < 0.005) by 71.5% in the presence of forskolin (10 microM), adenylyl cyclase activator, however, it was abolished (p < 0.001) by pretreatment with BAPTA-AM (25 microM), a membrane permeable Ca(2+) chelator. On the other hand, neither low Cl(2-) (75 mM, isotonic) nor hypertonicity (+NaCl or +mannitol, 500 mosmol kg H(2)O(-1)) could significantly increase the ATP release. Further, forskolin or ionomycin (a Ca(2+) ionophore) or, both, failed to stimulate ATP release under the isotonic condition. In conclusion, first, hypertonicity and changes in Cl(-) concentrations are not effective signals for the ATP release; second, hypotonicity-induced ATP release is potentiated by the level of intracellular Ca(2+) and cAMP; and third, a biphasic increase in ATP release and its low rate at the peak support the hypothesis that ATP is released through a non-conducting pathway model, such as exocytosis, or through a volume-dependent, ATP-conductive anion channel.     

Interleukin-9 and Interleukin-13 augment UTP-induced Cl ion transport via hCLCA1 expression in a human bronchial epithelial cell line

BACKGROUND: IL-9 and IL-13 induce airway goblet cell metaplasia, which is associated with expression of a Ca(2+)-activated Cl channel, hCLCA1. OBJECTIVE: As UTP stimulates both mucin secretion and Cl ion transport via a Ca(2+)-dependent pathway, the purpose of this study is to determine whether IL-9 and IL-13 affect UTP-induced Cl ion transport in human bronchial epithelial cell line 16HBE cells, and if they do, to elucidate whether such an effect is associated with hCLCA1 expression. METHODS: The increases in short-circuit current (I(sc)) in response to UTP were measured in the presence of amiloride by the Ussing chamber method. The morphology of epithelial cells was assessed by light microscopic findings, and hCLCA1 expression was investigated by immunocytochemistry and immunoblotting. RESULTS: UTP-induced increases in I(sc) in the cells treated with IL-9 or IL-13 for 48 h were greater than those in non-treated cells, and the potency of IL-13 was greater than that of IL-9. Pre-treatment with Ca(2+)-activated Cl channel inhibitors diisothocyanatostilbene-2, 2-disulphonic acid and niflumic acid completely inhibited the augmenting effects of IL-9 and IL-13 on I(sc). The epithelial layer of the cells treated with IL-9 or IL-13 was thicker than that of non-treated cells. The expression of hCLCA1 protein was induced by IL-13 in a concentration-dependent manner. These effects of IL-13 were more potent than those of IL-9. CONCLUSION: IL-9 and IL-13 augmented UTP-induced Cl ion transport, probably via proliferation of the cells with hCLCA1 expression, and IL-13 was more potent than IL-9 in producing such an effect in 16HBE cells.     

Effects of ouabain and amiloride on Na pathways in turtle bladders.

In a Na-rich bathing system, addition of amiloride to the mucosal fluid of turtle bladders produces decreased in the transepithelial potential difference (PD), short-circuiting current (I-sc), and conductance. Removal of amiloride results in complete reversal of these changes; and this reversibility is incomplete in amiloride-blocked bladders exposed to ouabain. In a Na-free bathing system, step increased in mucosal [Na] evoke rapid initial spikes in PD, Isc, and conductance, the magnitudes of which are independent of prior ouabain treatment. After these spikes, PD and Isc in the ouabain-treated bladder rapidly decay, while conductance remains unchanged and high. This unchanging conductance, plus the fact that ouabain inhibits half the microsomal (Na plus K). ATPase of this tissue within 1 min, suggests that ouabain inhibits Na pumping without changing tissue conductance. The delayed decrease in conductance (beginning 30 min after ouabain addition), a nonspecific and secondary effect of ouabain, is due to a concomitant collapse of the intercellular channels.