Cholinergic-induced anion secretion in murine jejunal enteroids involves synergy between muscarinic and nicotinic pathways

Acetylcholine induces robust electrogenic anion secretion in mammalian intestine and it has long been hypothesized that it mediates the epithelial response through the M3 and, to a lesser extent, the M1 muscarinic receptors in the mouse. However, nicotinic receptors have recently been identified in intestinal enterocytes by quantitative real-time (qRT)-PCR/RNAseq, although any direct influence on intestinal transport has not been identified. We tested the hypothesis that cholinergic-induced anion secretion in the intestine is a result of both muscarinic and nicotinic pathways that are intrinsic to the intestinal epithelia. We developed a method to generate mouse jejunal enteroid monolayers which were used to measure active electrogenic anion secretion by the Ussing chamber/voltage-clamp technique. Here, we show that the cholinergic agonist carbachol (CCh) and the muscarinic agonist bethanechol (BCh) stimulate short-lived, concentration-dependent anion secretion in the epithelial cell-only enteroid monolayers. The muscarinic antagonist atropine completely inhibited CCh- and BCh-induced secretion, while the nicotinic antagonist hexamethonium reduced the CCh response by ~45%. While nicotine alone did not alter anion secretion, it increased the BCh-induced increase in short-circuit current in a concentration-dependent manner; this synergy was prevented by pretreatment with hexamethonium. In addition to being sensitive to hexamethonium, monolayers express both classes of cholinergic receptor by qRT-PCR, including 13 of 16 nicotinic receptor subunits. Our findings indicate that an interaction between muscarinic and nicotinic agonists synergistically stimulates anion secretion in mouse jejunal epithelial cells and identify a role for epithelial nicotinic receptors in anion secretion.

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Regulation of electrolyte transport across cultured endometrial epithelial cells by prolactin

Journal of Endocrinology ◽

10.1677/joe-08-0077 ◽

2008 ◽

Vol 197 (3) ◽

pp. 575-582 ◽

Cited By ~ 9

Author(s):

Chatsri Deachapunya ◽

Sutthasinee Poonyachoti ◽

Nateetip Krishnamra

Keyword(s):

Epithelial Cells ◽

Short Circuit ◽

Short Circuit Current ◽

Disulfonic Acid ◽

Maximum Effect ◽

Channel Blockers ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Anion Secretion ◽

Endometrial Epithelial Cells

The effect of prolactin (PRL) on ion transport across the porcine glandular endometrial epithelial cells was studied in primary cell culture using the short-circuit current technique. Addition of 1 μg/ml PRL either to the apical solution or to the basolateral solution produced a peak followed by a sustained increase in Isc, but with a lesser response when PRL was added apically. Basolateral addition of PRL increased the Isc in a concentration-dependent manner with a maximum effect at 1 μg/ml and an effective concentration value of 120 ng/ml. The PRL-stimulated Isc was significantly reduced by pretreatment with an apical addition of 5-nitro-2-(3-phenylpropylamino) benzoic acid (200 μM), diphenylamine-2-carboxylic acid (1 mM) or 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (200 μM), Cl− channel blockers, but not by amiloride (10 μM), a Na+ channel blocker. In addition, pretreatment with bumetanide (200 μM), a Na+–K+–2Cl− cotransporter inhibitor, in the basolateral solution significantly reduced the PRL-stimulated Isc. Replacement of Cl− or in the bathing solutions also decreased the Isc response to PRL. Pretreatment of the monolayer with AG490 (50 μM), an inhibitor of JAK2 activity significantly inhibited the PRL-induced increase in Isc. Western blot analysis of the porcine endometrial epithelial cells revealed the presence of short isoform of PRL receptor (PRLR-S) that could be regulated by 17β-estradiol. The results of this investigation showed that PRL acutely stimulated anion secretion across the porcine endometrial epithelial cells possibly through PRLR-S present in both apical and basolateral membranes. The PRL response appeared to be mediated by the JAK2-dependent pathway.

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Desensitization of P2Y2receptor-activated transepithelial anion secretion

AJP Cell Physiology ◽

10.1152/ajpcell.1999.276.4.c777 ◽

1999 ◽

Vol 276 (4) ◽

pp. C777-C787 ◽

Cited By ~ 37

Author(s):

Lane L. Clarke ◽

Matthew C. Harline ◽

Miguel A. Otero ◽

Geraldine G. Glover ◽

Richard C. Garrad ◽

...

Keyword(s):

Receptor Agonist ◽

Inositol Phosphate ◽

Short Circuit ◽

Short Circuit Current ◽

Extracellular Nucleotides ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Anion Secretion ◽

Ussing Chambers ◽

Intracellular Ca

Desensitization of P2Y2 receptor-activated anion secretion may limit the usefulness of extracellular nucleotides in secretagogue therapy of epithelial diseases, e.g., cystic fibrosis (CF). To investigate the desensitization process for endogenous P2Y2 receptors, freshly excised or cultured murine gallbladder epithelia (MGEP) were mounted in Ussing chambers to measure short-circuit current ( I sc), an index of electrogenic anion secretion. Luminal treatment with nucleotide receptor agonists increased the I sc with a potency profile of ATP = UTP > 2-methylthioATP >> α,β-methylene-ATP. RT-PCR revealed the expression of P2Y2 receptor mRNA in the MGEP cells. The desensitization of anion secretion required a 10-min preincubation with the P2Y2receptor agonist UTP and increased in a concentration-dependent manner (IC50 ≈ 10−6 M). Approximately 40% of the anion secretory response was unaffected by maximal desensitizing concentrations of UTP. Recovery from UTP-induced desensitization was rapid (<10 min) at preincubation concentrations less than the EC50 (1.9 × 10−6 M) but required progressively longer time periods at greater concentrations. UTP-induced total inositol phosphate production and intracellular Ca2+ mobilization desensitized with a concentration dependence similar to that of anion secretion. In contrast, maximal anion secretion induced by Ca2+ ionophore ionomycin was unaffected by preincubation with a desensitizing concentration of UTP. It was concluded that 1) desensitization of transepithelial anion secretion stimulated by the P2Y2 receptor agonist UTP is time and concentration dependent; 2) recovery from desensitization is prolonged (>90 min) at UTP concentrations >10−5 M; and 3) UTP-induced desensitization occurs before the operation of the anion secretory mechanism.

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Human colonic epithelial cells express galanin-1 receptors, which when activated cause Cl− secretion

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1999.276.1.g64 ◽

1999 ◽

Vol 276 (1) ◽

pp. G64-G72 ◽

Cited By ~ 5

Author(s):

Richard V. Benya ◽

Jorge A. Marrero ◽

Denis A. Ostrovskiy ◽

Athanasia Koutsouris ◽

Gail Hecht

Keyword(s):

Short Circuit ◽

Ussing Chamber ◽

Short Circuit Current ◽

Peptide Hormone ◽

Human Colon ◽

Smooth Muscle Contraction ◽

Dependent Manner ◽

Gi Tract ◽

T84 Cells ◽

Concentration Dependent Manner

Galanin is a peptide hormone widely expressed in the central nervous system and gastrointestinal (GI) tract. Within the GI tract galanin is present in enteric nerve terminals where it is known to modulate intestinal motility by altering smooth muscle contraction. Recent studies also show that galanin can alter intestinal short-circuit current ( I sc) but with differing results observed in rats, rabbits, guinea pigs, and pigs. In contrast, nothing is known about the ability of galanin to alter ion transport in human intestinal epithelial tissues. By RT-PCR, we determined that these tissues express only the galanin-1 receptor (Gal1-R) subtype. To evaluate Gal1-R pharmacology and physiology, we studied T84 cells. Gal1-R expressed by these cells bound galanin rapidly (half time 1–2 min) and with high affinity (inhibitor constant 0.7 ± 0.2 nM). T84 cells were then studied in a modified Ussing chamber and alterations in I sc, a measure of all ion movement across the tissue, were determined. Maximal increases in I sc were observed in a concentration-dependent manner around 2 min after stimulation with peptide, with 1 μM galanin causing I sc to rise more than eightfold and return to baseline occurring within 10 min. The increase in galanin-induced I sc was shown by125I efflux studies to be due to Cl− secretion, which occurred independently of alterations in cAMP and phospholipase C. Rather, Cl− secretion is mediated via a Ca2+-dependent, pertussis toxin-sensitive mechanism. These data suggest that galanin released by enteric nerves may act as a secretagogue in the human colon by activating Gal1-R.

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Enterotoxigenicity of Mature 45-Kilodalton and Processed 35-Kilodalton Forms of Hemagglutinin Protease Purified from a Cholera Toxin Gene-Negative Vibrio cholerae Non-O1, Non-O139 Strain

Infection and Immunity ◽

10.1128/iai.74.5.2937-2946.2006 ◽

2006 ◽

Vol 74 (5) ◽

pp. 2937-2946 ◽

Cited By ~ 27

Author(s):

A. Ghosh ◽

D. R. Saha ◽

K. M. Hoque ◽

M. Asakuna ◽

S. Yamasaki ◽

...

Keyword(s):

Cholera Toxin ◽

Vibrio Cholerae ◽

Hela Cells ◽

Histopathological Examination ◽

Short Circuit ◽

Ussing Chamber ◽

Short Circuit Current ◽

Toxin Gene ◽

Dependent Manner ◽

A Cell

ABSTRACT Cholera toxin gene-negative Vibrio cholerae non-O1, non-O139 strain PL-21 is the etiologic agent of cholera-like syndrome. Hemagglutinin protease (HAP) is one of the major secretory proteins of PL-21. The mature 45-kDa and processed 35-kDa forms of HAP were purified in the presence and absence of EDTA from culture supernatants of PL-21. Enterotoxigenicities of both forms of HAP were tested in rabbit ileal loop (RIL), Ussing chamber, and tissue culture assays. The 35-kDa HAP showed hemorrhagic fluid response in a dose-dependent manner in the RIL assay. Histopathological examination of 20 μg of purified protease-treated rabbit ileum showed the presence of erythrocytes and neutrophils in the upper part of the villous lamina propria. Treatment with 40 μg of protease resulted in gross damage of the villous epithelium with inflammation, hemorrhage, and necrosis. The 35-kDa form of HAP, when added to the lumenal surface of rat ileum loaded in an Ussing chamber, showed a decrease in the intestinal short-circuit current and a cell rounding effect on HeLa cells. The mature 45-kDa form of HAP showed an increase in intestinal short-circuit current in an Ussing chamber and a cell distending effect on HeLa cells. These results show that HAP may play a role in the pathogenesis of PL-21.

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Protein kinase C potentiates cAMP-stimulated mouse duodenal mucosal bicarbonate secretion in vitro

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00251.2003 ◽

2004 ◽

Vol 286 (5) ◽

pp. G814-G821 ◽

Cited By ~ 10

Author(s):

Bi-Guang Tuo ◽

Jimmy Y. C. Chow ◽

Kim E. Barrett ◽

Jon I. Isenberg

Keyword(s):

Short Circuit ◽

Short Circuit Current ◽

Duodenal Mucosa ◽

Bicarbonate Secretion ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Ussing Chambers ◽

Duodenal Bicarbonate Secretion

PKC has been shown to regulate epithelial Cl- secretion in a variety of models. However, the role of PKC in duodenal mucosal bicarbonate secretion is less clear. We aimed to investigate the role of PKC in regulation of duodenal mucosal bicarbonate secretion. Bicarbonate secretion by murine duodenal mucosa was examined in vitro in Ussing chambers using a pH-stat technique. PKC isoform expression and activity were assessed by Western blotting and in vitro kinase assays, respectively. PMA (an activator of PKC) alone had no effect on duodenal bicarbonate secretion or short-circuit current ( Isc). When PMA and dibutyryl-cAMP (db-cAMP) were added simultaneously, PMA failed to alter db-cAMP-stimulated duodenal bicarbonate secretion or Isc ( P > 0.05). However, a 1-h preincubation with PMA potentiated db-cAMP-stimulated duodenal bicarbonate secretion and Isc in a concentration-dependent manner (from 10-8 to 10-5M) ( P < 0.05). PMA preincubation had no effects on carbachol- or heat-stable toxin-stimulated bicarbonate secretion. Western blot analysis revealed that PKCα, -γ, -ϵ, -θ, -μ, and -ι/λ were expressed in murine duodenal mucosa. Ro 31–8220 (an inhibitor active against PKCϵ, -α, -β, and -γ), but not Gö 6983 (an inhibitor active against PKCα, -γ, -β, and -δ), reversed the potentiating effect of PMA on db-cAMP-stimulated bicarbonate secretion. PMA also time- and concentration-dependently increased the activity of PKCϵ, an effect that was prevented by Ro 31–8220 but not Gö 6983. These results demonstrate that activation of PKC potentiates cAMP-stimulated duodenal bicarbonate secretion, whereas it does not modify basal secretion. The effect of PKC on cAMP-stimulated bicarbonate secretion is mediated by the PKCϵ isoform.

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Apical adenosine regulates basolateral Ca2+-activated potassium channels in human airway Calu-3 epithelial cells

AJP Cell Physiology ◽

10.1152/ajpcell.00556.2007 ◽

2008 ◽

Vol 294 (6) ◽

pp. C1443-C1453 ◽

Cited By ~ 14

Author(s):

Dong Wang ◽

Ying Sun ◽

Wei Zhang ◽

Pingbo Huang

Keyword(s):

Epithelial Cells ◽

Adenosine Receptors ◽

Short Circuit ◽

Ussing Chamber ◽

Specific Inhibitor ◽

Short Circuit Current ◽

Airway Epithelial Cells ◽

Intracellular Camp ◽

Anion Secretion ◽

Human Airway

In airway epithelial cells, apical adenosine regulates transepithelial anion secretion by activation of apical cystic fibrosis transmembrane conductance regulator (CFTR) via adenosine receptors and cAMP/PKA signaling. However, the potent stimulation of anion secretion by adenosine is not correlated with its modest intracellular cAMP elevation, and these uncorrelated efficacies have led to the speculation that additional signaling pathways may be involved. Here, we showed that mucosal adenosine-induced anion secretion, measured by short-circuit current ( Isc), was inhibited by the PLC-specific inhibitor U-73122 in the human airway submucosal cell line Calu-3. In addition, the Isc was suppressed by BAPTA-AM (a Ca2+ chelator) and 2-aminoethoxydiphenyl borate (2-APB; an inositol 1,4,5-trisphosphate receptor blocker), but not by PKC inhibitors, suggesting the involvement of PKC-independent PLC/Ca2+ signaling. Ussing chamber and patch-clamp studies indicated that the adenosine-induced PLC/Ca2+ signaling stimulated basolateral Ca2+-activated potassium (KCa) channels predominantly via A2B adenosine receptors and contributed substantially to the anion secretion. Thus, our data suggest that apical adenosine activates contralateral K+ channels via PLC/Ca2+ and thereby increases the driving force for transepithelial anion secretion, synergizing with its modulation of ipsilateral CFTR via cAMP/PKA. Furthermore, the dual activation of CFTR and KCa channels by apical adenosine resulted in a mixed secretion of chloride and bicarbonate, which may alter the anion composition in the secretion induced by secretagogues that elicit extracellular ATP/adenosine release. Our findings provide novel mechanistic insights into the regulation of anion section by adenosine, a key player in the airway surface liquid homeostasis and mucociliary clearance.

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NH2-terminal modification of a channel-forming peptide increases capacity for epithelial anion secretion

AJP Cell Physiology ◽

10.1152/ajpcell.2001.280.3.c451 ◽

2001 ◽

Vol 280 (3) ◽

pp. C451-C458 ◽

Cited By ~ 22

Author(s):

James R. Broughman ◽

Kathy E. Mitchell ◽

Roger L. Sedlacek ◽

Takeo Iwamoto ◽

John M. Tomich ◽

...

Keyword(s):

Therapeutic Potential ◽

Short Circuit ◽

Ussing Chamber ◽

Glycine Receptor ◽

Short Circuit Current ◽

Disulfonic Acid ◽

Α Subunit ◽

Anion Secretion ◽

Anion Conductance ◽

Selective Channel

A synthetic, channel-forming peptide, derived from the α-subunit of the glycine receptor (M2GlyR), has been synthesized and modified by adding four lysine residues to the NH2 terminus (N-K4-M2GlyR). In Ussing chamber experiments, apical N-K4-M2GlyR (250 μM) increased transepithelial short-circuit current ( I sc) by 7.7 ± 1.7 and 10.6 ± 0.9 μA/cm2 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-K4-M2GlyR caused a concentration-dependent increase in I sc ( k [1/2] = 190 μM) that was potentiated two- to threefold by 1-ethyl-2-benzimidazolinone. N-K4-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-K4-M2GlyR-mediated I sc. Whole cell membrane patch voltage-clamp studies revealed an N-K4-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-K4-M2GlyR forms a 50-pS anion conductance with a k [1/2] for Cl−of 290 meq. These results indicate that N-K4-M2GlyR forms an anion-selective channel in epithelial monolayers and shows therapeutic potential for the treatment of hyposecretory disorders such as cystic fibrosis.

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Histamine stimulates ion transport by dog pancreatic duct epithelial cells through H1receptors

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1998.275.1.g76 ◽

1998 ◽

Vol 275 (1) ◽

pp. G76-G84 ◽

Cited By ~ 6

Author(s):

Toan D. Nguyen ◽

Charles N. Okolo ◽

Mark W. Moody

Keyword(s):

Epithelial Cells ◽

Ion Transport ◽

Receptor Antagonist ◽

Pancreatic Duct ◽

Direct Action ◽

Short Circuit ◽

Short Circuit Current ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Ussing Chambers

Histamine affects pancreatic secretion, but its direct action on ion transport by pancreatic duct epithelial cells (PDEC) has not been defined. We now characterize the secretory effects of histamine on cultured, well-differentiated, and nontransformed dog PDEC. Histamine stimulated, in a concentration-dependent manner (1–100 μM), a cellular125I−efflux that was inhibited by 500 μM 5-nitro-2-(3-phenylpropylamino)benzoic acid, 2.5 mM diphenylamine-2-carboxylate, and 500 μM DIDS and thus mediated through Ca2+-activated Cl− channels. Histamine-stimulated125I−efflux was 1) inhibited by 100 μM diphenhydramine, an H1receptor antagonist, 2) resistant to 1 mM cimetidine, an H2 receptor antagonist, 3) not reproduced by 1 mM dimaprit, an H2 agonist, and 4) inhibited by 50 μM 1,2-bis(2-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid-AM, a Ca2+ chelator, suggesting that it was mediated through H1 receptors acting via increased cytosolic Ca2+. Histamine also stimulated a86Rb+efflux that was sensitive to 100 nM charybdotoxin and thus mediated through Ca2+-activated K+ channels. When PDEC monolayers were studied in Ussing chambers, a short-circuit current of 21.7 ± 3.1 μA/cm2 was stimulated by 100 μM histamine. This effect was inhibited by diphenhydramine but not cimetidine, was not reproduced with dimaprit, and was observed only after serosal addition of histamine, suggesting that it was mediated by basolateral H1 receptors on PDEC. In conclusion, histamine, acting through basolateral H1 receptors, activates both Ca2+-activated Cl− and K+ channels; in this manner, it may regulate PDEC secretion in normal or inflamed pancreas.

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SARS-COV-2 induced Diarrhea is inflammatory, Ca2+ Dependent and involves activation of calcium activated Cl channels

10.1101/2021.04.27.441695 ◽

2021 ◽

Author(s):

Mark Donowitz ◽

Chung-Ming Tse ◽

Karol Dokladny ◽

Manmeet Rawat ◽

Ivy Horwitz ◽

...

Keyword(s):

Short Circuit ◽

Ussing Chamber ◽

Short Circuit Current ◽

Proximal Colon ◽

K Channel ◽

Mrna Levels ◽

Apical Surface ◽

Live Virus ◽

Anion Secretion ◽

Calcium Buffer

ABSTRACTDiarrhea occurs in 2-50% of cases of COVID-19 (∼8% is average across series). The diarrhea does not appear to account for the disease mortality and its contribution to the morbidity has not been defined, even though it is a component of Long Covid or post-infectious aspects of the disease. Even less is known about the pathophysiologic mechanism of the diarrhea. To begin to understand the pathophysiology of COVID-19 diarrhea, we exposed human enteroid monolayers obtained from five healthy subjects and made from duodenum, jejunum, and proximal colon to live SARS-CoV-2 and virus like particles (VLPs) made from exosomes expressing SARS-CoV-2 structural proteins (Spike, Nucleocapsid, Membrane and Envelope). Results: 1) Live virus was exposed apically for 90 min, then washed out and studied 2 and 5 days later. SARS-Cov-2 was taken up by enteroids and live virus was present in lysates and in the apical>>basolateral media of polarized enteroids 48 h after exposure. This is the first demonstration of basolateral appearance of live virus after apical exposure. High vRNA concentration was detected in cell lysates and in the apical and basolateral media up to 5 days after exposure. 2) Two days after viral exposure, cytokine measurements of media showed significantly increased levels of IL-6, IL-8 and MCP-1. 3) Two days after viral exposure, mRNA levels of ACE2, NHE3 and DRA were reduced but there was no change in mRNA of CFTR. NHE3 protein was also decreased. 4) Live viral studies were mimicked by some studies with VLP exposure for 48 h. VLPs with Spike-D614G bound to the enteroid apical surface and was taken up; this resulted in decreased mRNA levels of ACE2, NHE3, DRA and CFTR. 4) VLP effects were determined on active anion secretion measured with the Ussing chamber/voltage clamp technique. S-D614G acutely exposed to apical surface of human ileal enteroids did not alter the short-circuit current (Isc). However, VLPS-D614G exposure to enteroids that were pretreated for ∼24 h with IL-6 plus IL-8 induced a concentration dependent increase in Isc indicating stimulated anion secretion, that was delayed in onset by ∼8 min. The anion secretion was inhibited by apical exposure to a specific calcium activated Cl channel (CaCC) inhibitor (AO1) but not by a specific CFTR inhibitor (BP027); was inhibited by basolateral exposure to the K channel inhibit clortimazole; and was prevented by pretreatment with the calcium buffer BAPTA-AM. 5) The calcium dependence of the VLP-induced increase in Isc was studied in Caco-2/BBe cells stably expressing the genetically encoded Ca2+ sensor GCaMP6s. 24 h pretreatment with IL-6/IL-8 did not alter intracellular Ca2+. However, in IL-6/IL-8 pretreated cells, VLP S-D614G caused appearance of Ca2+waves and an overall increase in intracellular Ca2+ with a delay of ∼10 min after VLP addition. We conclude that the diarrhea of COVID-19 appears to an example of a calcium dependent inflammatory diarrhea that involves both acutely stimulated Ca2+ dependent anion secretion (stimulated Isc) that involves CaCC and likely inhibition of neutral NaCl absorption (decreased NHE3 protein and mRNA and decreased DRA mRNA).

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Asymmetric effects of H2O2 on alveolar epithelial barrier properties

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1993.264.3.l308 ◽

1993 ◽

Vol 264 (3) ◽

pp. L308-L315 ◽

Cited By ~ 7

Author(s):

K. J. Kim ◽

D. J. Suh

Keyword(s):

Alveolar Epithelial Cell ◽

Short Circuit ◽

Ussing Chamber ◽

Short Circuit Current ◽

Barrier Properties ◽

Ringer Solution ◽

Alveolar Epithelial Cells ◽

Dependent Manner ◽

Polycarbonate Membrane ◽

Alveolar Epithelial

The effects of H2O2 on active ion transport and resistance to passive solute flow were studied utilizing rat alveolar epithelial cell monolayers cultured on permeable supports. Type II alveolar epithelial cells were plated onto tissue culture-treated polycarbonate membrane filters. The resulting confluent monolayers on days 3 and 4 were mounted in a modified Ussing chamber and bathed on both sides with N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-buffered Ringer solution. These monolayers have a high transepithelial resistance (> 2,000 omega.cm2) and actively transport Na+ from apical fluid. H2O2 (0-100 mM) was then delivered to either apical or basolateral fluid. The changes in short-circuit current (Isc) and monolayer resistance (R) in response to the exogenous hydroperoxide were measured. To determine the degree of cellular catalase participation in protection against H2O2 injury to the barrier, experiments were repeated in the presence of 20 mM aminotriazole (ATAZ; an inhibitor of catalase) in the same bathing fluid as the hydroperoxide. Results indicated that H2O2 decreased Isc and R gradually in a dose-dependent manner. The effective concentration of apical H2O2 at which Isc (or R) was decreased by 50% at 1 h (ED50) was approximately 4 mM. However, basolateral H2O2 exposure led to ED50 for Isc (and R) of approximately 0.04 mM. Inhibition of cellular catalase yielded ED50 for Isc (and R) of approximately 0.4 mM when H2O2 was given apically, while ED50 for basolateral exposure to H2O2 did not change in the presence of ATAZ. The rate of H2O2 consumption in apical and basolateral bathing fluids was the same, while cellular catalase activity rose gradually with time in culture.(ABSTRACT TRUNCATED AT 250 WORDS)

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