Calcium-dependent chloride secretion across cultures of human tracheal surface epithelium and glands

1993 ◽  
Vol 265 (2) ◽  
pp. L170-L177 ◽  
Author(s):  
M. Yamaya ◽  
T. Ohrui ◽  
W. E. Finkbeiner ◽  
J. H. Widdicombe
Keyword(s):  
Time Course ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Cell Types ◽  
Chloride Secretion ◽  
Surface Epithelium ◽  
Acetoxymethyl Ester ◽  
Calcium Dependent ◽  
Gland Cells ◽  
Active Ion Transport

Surface epithelium and gland cells from human trachea were cultured on porous-bottom inserts and loaded with fura 2 to permit measurement of the intracellular calcium concentration ([Ca2+]i). Short-circuit current (Isc), an index of transepithelial active ion transport, was measured on cells from the same cultures. Surface epithelial [Ca2+]i of 82 +/- 15 nM was increased transiently by isoproterenol, histamine, and bradykinin with maximal increases of 88 +/- 17, 480 +/- 149, and 978 +/- 214 nM (n = 15), respectively. Baseline [Ca2+]i in cultured gland cells of 68 +/- 11 nM was increased transiently by isoproterenol, histamine, methacholine, and bradykinin with maximal increases of 105 +/- 19, 233 +/- 47, 327 +/- 121, and 634 +/- 151 nM (n = 17-21), respectively. In both cell types, mediators that increased [Ca2+]i also increased Isc with a time course identical to the increase in [Ca2+]i. Pretreatment with the calcium chelator, 1,2-bis-(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid, acetoxymethyl ester (BAPTA-AM), had no effect on basal Isc or transepithelial resistance but markedly inhibited both the Isc and [Ca2+]i responses to agonists. Forskolin (10(-5) M), 3-isobutyl-1-methylxanthine (10(-3) M), dibutyryl adenosine 3',5'-cyclic monophosphate (10(-3) M), and 8-(4-chlorophenylthio)-cAMP (10(-3) M) had no or only trivial effects on Isc and [Ca2+]i. We suggest that mediators increase Isc across human airway epithelium by activating Ca-dependent basolateral K channels, resulting in hyperpolarization and an increased driving force for Cl exit through apical membrane Cl channels.

Role of CFTR in chloride secretion across human tracheal epithelium

1995 ◽  
Vol 269 (5) ◽  
pp. L561-L566 ◽  
Author(s):  
B. Q. Shen ◽  
R. J. Mrsny ◽  
W. E. Finkbeiner ◽  
J. H. Widdicombe
Keyword(s):  
Apical Membrane ◽  
Short Circuit ◽  
Primary Cultures ◽  
Short Circuit Current ◽  
Chloride Secretion ◽  
Culture Conditions ◽  
Tracheal Epithelium ◽  
Disulfonic Acid ◽  
Acetoxymethyl Ester ◽  
Cl Channels

We have tested two hypotheses: 1) the cystic fibrosis transmembrane conductance regulator (CFTR) represents the predominant Cl conductance in the apical membrane of human tracheal epithelium, and 2) CFTR in this tissue is close to maximally activated under baseline conditions. In support of the first hypothesis, we found 1) when the level of differentiation of cultures was varied by varying the culture conditions, there was a significant positive correlation between the levels of CFTR and the magnitude of mediator-induced Cl secretion. 2) Amiloride-insensitive baseline short-circuit current (Isc) and mediator-induced increases in Isc were inhibited by diphenylamine-2-carboxylic acid (DPAC) but not by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), a pharmacology consistent with passage of apical membrane Cl current through CFTR; Ca-activated Cl channels are inhibited by DIDS but not by DPAC. 3) Raising temperature from 22 degrees to 37 degrees C increased 125I efflux, and this increase was inhibited by DPAC and blockers of protein kinase A, but not by DIDS or 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester. In support of the second hypothesis, we have earlier shown [M. Yamaya, W.E. Finkbeiner, S.Y. Chun, and J.H. Widdicombe. Am. J. Physiol. 262 (Lung Cell. Mol. Physiol. 6): L713-L724, 1992] that adenosine 3',5'-cyclic monophosphate (cAMP)-elevating agents are essentially without effect on Isc across primary cultures of human tracheal epithelium. Here, we further show that these agents are also usually without effect on 125I efflux; the mean increase in efflux in response to elevating cAMP was approximately 20% that of raising temperature from 22 degrees to 37 degrees C.

Neuropeptide Y inhibits chloride secretion in the shark rectal gland

1993 ◽  
Vol 265 (2) ◽  
pp. R439-R446 ◽  
Author(s):  
P. Silva ◽  
F. H. Epstein ◽  
K. J. Karnaky ◽  
S. Reichlin ◽  
J. N. Forrest
Keyword(s):  
Neuropeptide Y ◽  
Direct Action ◽  
Short Circuit ◽  
Primary Cultures ◽  
Short Circuit Current ◽  
Chloride Secretion ◽  
Rectal Gland ◽  
Maximal Effect ◽  
Gland Cells ◽  
Shark Rectal Gland

We studied the effects of the 36-amino acid peptide, neuropeptide Y (NPY), on salt secretion by the rectal gland of Squalus acanthias. We used three preparations: whole isolated perfused glands, freshly prepared separated rectal gland tubules, and confluent monolayers of cultured rectal gland cells. In perfused glands NPY inhibited secretion stimulated by vasoactive intestinal peptide (VIP), forskolin, or adenosine 3',5'-cyclic monophosphate (cAMP) and theophylline. Maximal inhibition of 63 +/- 3.4% was seen at 3 x 10(-8) M NPY, with half-maximal effect at 3 x 10(-9) M. NPY did not inhibit the basal activity of rectal gland adenylate cyclase or that stimulated by VIP. The inhibitory action of NPY was not prevented by procaine, nifedipine, or diltiazem, suggesting that it was not secondary to the release of somatostatin or other unknown neurotransmitters from rectal gland nerves. In confirmation, somatostatin was not detected in the venous effluent after administration of NPY. NPY also inhibited transport-related oxygen consumption in separated rectal gland tubules and inhibited short-circuit current generated by confluent monolayers of primary cultures of rectal gland cells. The results indicate that NPY inhibits chloride secretion by a direct action on cells of the shark rectal gland at a site distal to the generation of cAMP.

Ion transport and structure of urinary bladder epithelium of Amphiuma

1976 ◽  
Vol 231 (2) ◽  
pp. 501-508 ◽  
Author(s):  
TL Mullen ◽  
M Kashgarian ◽  
D Biemesderfer ◽  
GH Giebisch ◽  
TU Biber
Keyword(s):  
Urinary Bladder ◽  
Short Circuit ◽  
Electrical Potential ◽  
Short Circuit Current ◽  
Cell Types ◽  
Surface Epithelium ◽  
Electrical Potential Difference ◽  
Basal Cells ◽  
Bladder Epithelium

The urinary bladder of Amphiuma exhibits stable transport properties and an electrical potential difference in vitro. The lumen is significantly negative to the serosa and under short-circuited conditions flux rations for Na and Cl of 5.92 +/- 0.42 and 1.81 +/- 0.20, respectively, were observed. The close agreement between the short-circuit current and net Na flux suggests that most, if not all, of the current is carried by Na. Both ouabain and amiloride decreased the short-circuit current and the mucosal-to-serosal (M leads to S) flux of Na. Furosemide caused a transient increase in the M leads to S flux of Na and Cl but ADH was without effect. In bladders that had high transmural resistance, a net movement of K in the M leads to S direction under short-circuited conditions with flux ratios of up to 2 could be observed. The epithelium of the Amphiuma bladder consists of three cell types: granular cells, basal cells, and mitochondria-rich cells. No goblet cells are present. The mitochondria-rich cells comprise less than 5% of the population of the surface epithelium in Amphiuma in contrast to other amphibian bladders, where it accounts for up to 25% of the population.

Dual effects of a phorbol ester on calcium-dependent chloride secretion by T84 epithelial cells

1992 ◽  
Vol 262 (1) ◽  
pp. C15-C22 ◽  
Author(s):  
U. Kachintorn ◽  
P. Vongkovit ◽  
M. Vajanaphanich ◽  
S. Dinh ◽  
K. E. Barrett ◽  
...  
Keyword(s):  
Short Circuit ◽  
Short Circuit Current ◽  
Cytosolic Calcium ◽  
Chloride Secretion ◽  
Inhibitory Effect ◽  
K Channel ◽  
Transient Effects ◽  
Stimulatory Action ◽  
Cl Secretion ◽  
Calcium Dependent

Ca(2+)-dependent secretagogues (e.g., carbachol, histamine, ionomycin, and 4-bromo-A23187) have relatively transient effects on chloride secretion, even if there is a sustained increase in cytosolic calcium ([Ca2+]i) (as for the ionophores). Because these agents increase both [Ca2+]i and protein kinase C (PKC) activity, chloride secretion might be stimulated by [Ca2+]i and terminated by PKC activity. We tested the effect of a PKC activator, phorbol 12-myristate 13-acetate (PMA), on Cl- secretion by T84 cell monolayers by measuring short-circuit current (Isc). PMA alone had no effect on Isc but potentiated increases in Isc when added 10 min or less before Ca(2+)-dependent secretagogues. Chelation of [Ca2+]i with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid inhibited the increases both in [Ca2+]i and Isc induced by carbachol with or without brief PMA pretreatment. Longer preincubations with PMA inhibited Isc responses to Ca(2+)-dependent secretagogues, even when increased [Ca2+]i was sustained by ionophores. Inhibitors of PKC could reverse the inhibitory effect of PMA but did not reverse the potentiating effect. The effects of PMA on Cl- secretion were reproduced by 1,2-dioctanoyl-sn-glycerol and were mirrored by effects on K+ channel opening. Thus PMA has dual effects on chloride secretion. Initially, it exerts a stimulatory action and subsequently an inhibitory action. The stimulatory effect only occurs if Ca(2+)-dependent secretion is ongoing. The inhibitory effect of PMA is mediated by PKC and cannot be overcome by increasing [Ca2+]i.

Normalization of ion transport in murine cystic fibrosis nasal epithelium using gene transfer

1997 ◽  
Vol 273 (2) ◽  
pp. C734-C740 ◽  
Author(s):  
L. J. MacVinish ◽  
C. Goddard ◽  
W. H. Colledge ◽  
C. F. Higgins ◽  
M. J. Evans ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Chloride Channels ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Chloride Secretion ◽  
Nasal Epithelium ◽  
Sodium Absorption ◽  
Calcium Dependent ◽  
Cf Transmembrane Conductance Regulator ◽  
Therapy Studies

The murine nasal epithelium was investigated by the short-circuit current (SCC) technique. Electrogenic sodium absorption was revealed by addition of amiloride and calcium-dependent chloride secretion by the addition of amiloride and calcium-dependent chloride secretion by the addition of 2,5-di-(tert-butyl)-1,4-benzohydroquinone (TBHQ)/ionomycin. In the presence of these agents a further increase in SCC was obtained by addition of forskolin. Epithelia from both cystic fibrosis (CF) null (Cftrtm1Cam) and CF delta F508 (Cftrtm2Cam) mice had enhanced sodium absorption compared with controls, whereas only delta F508 epithelia had increased calcium-dependent chloride secretion. Both strains gave nasal epithelia that showed significantly reduced responses to forskolin, due to the absence of CF transmembrane conductance regulator (CFTR) chloride channels. In Cftrtm2Cam nasal epithelia the forskolin responses were not significantly different from zero. Transfection of these mice with the plasmid pTRIAL10-CFTR2 complexed with cationic liposomes normalized the transporting activity in the nasal epithelium. Basal SCC and calcium-dependent chloride secretion were significantly reduced, whereas CFTR-dependent chloride secretion was increased to normal values. Amiloride-sensitive SCC was reduced by transfection but failed to reach significance. The similarity of murine CF nasal epithelium to that in human CF airways makes the model valuable for gene therapy studies.

Endothelin stimulates chloride secretion across canine tracheal epithelium

1991 ◽  
Vol 261 (2) ◽  
pp. L188-L194 ◽  
Author(s):  
P. I. Plews ◽  
Z. A. Abdel-Malek ◽  
C. A. Doupnik ◽  
G. D. Leikauf
Keyword(s):  
Epithelial Cells ◽  
Short Circuit ◽  
Second Messengers ◽  
Short Circuit Current ◽  
Chloride Secretion ◽  
Tracheal Epithelium ◽  
Membrane Phospholipids ◽  
Cl Secretion ◽  
Tracheal Epithelial Cells ◽  
Tracheal Epithelial

The endothelins (ET) are a group of isopeptides produced by a number of cells, including canine tracheal epithelial cells. Because these compounds are endogenous peptides that may activate eicosanoid metabolism, we investigated the effects of ET on Cl secretion in canine tracheal epithelium. Endothelin 1 (ET-1) was found to produce a dose-dependent change in short-circuit current (Isc) that increased slowly and reached a maximal value within 10-15 min. When isopeptides of ET were compared, 300 nM ET-1 and ET-2 produced comparable maximal increases in Isc, whereas ET-3 produced smaller changes in Isc (half-maximal concentrations of 2.2, 7.2, and 10.4 nM, respectively). Ionic substitution of Cl with nontransported anions, iodide and gluconate, reduced ET-1-induced changes in Isc. Furthermore, the response was inhibited by the NaCl cotransport inhibitor, furosemide. In paired tissues, ET-1 significantly increased mucosal net 36Cl flux without significant effect on 22Na flux. The increase in Isc induced by ET was diminished by pretreatment with indomethacin. The second messengers mediating the increase in Isc were investigated in cultured canine tracheal epithelial cells. ET-1 stimulated the release of [3H]arachidonate from membrane phospholipids, increased intracellular Ca2+ (occasionally producing oscillations), and increased adenosine 3',5'-cyclic monophosphate accumulation. The latter was diminished by indomethacin. Thus ET is a potent agonist of Cl secretion (with the isopeptides having the following potency: ET-1 greater than or equal to ET-2 greater than ET-3) and acts, in part, through a cyclooxygenase-dependent mechanism.

Basolateral Cl− uptake mechanisms in Xenopus laevis lung epithelium

2010 ◽  
Vol 299 (1) ◽  
pp. R92-R100 ◽  
Author(s):  
Jens Berger ◽  
Martin Hardt ◽  
Wolfgang G. Clauss ◽  
Martin Fronius
Keyword(s):  
Xenopus Laevis ◽  
Ion Transport ◽  
Anion Exchanger ◽  
Short Circuit ◽  
Ussing Chamber ◽  
Short Circuit Current ◽  
Transport Processes ◽  
Lung Epithelium ◽  
Active Ion Transport ◽  
Uptake Mechanisms

A thin liquid layer covers the lungs of air-breathing vertebrates. Active ion transport processes via the pulmonary epithelial cells regulate the maintenance of this layer. This study focuses on basolateral Cl− uptake mechanisms in native lungs of Xenopus laevis and the involvement of the Na+/K+/2 Cl− cotransporter (NKCC) and HCO3−/Cl− anion exchanger (AE), in particular. Western blot analysis and immunofluorescence staining revealed the expression of the NKCC protein in the Xenopus lung. Ussing chamber experiments demonstrated that the NKCC inhibitors (bumetanide and furosemide) were ineffective at blocking the cotransporter under basal conditions, as well as under pharmacologically stimulated Cl−-secreting conditions (forskolin and chlorzoxazone application). However, functional evidence for the NKCC was detected by generating a transepithelial Cl− gradient. Further, we were interested in the involvement of the HCO3−/Cl− anion exchanger to transepithelial ion transport processes. Basolateral application of DIDS, an inhibitor of the AE, resulted in a significantly decreased the short-circuit current (ISC). The effect of DIDS was diminished by acetazolamide and reduced by increased external HCO3− concentrations. Cl− secretion induced by forskolin was decreased by DIDS, but this effect was abolished in the presence of HCO3−. These experiments indicate that the AE at least partially contributes to Cl− secretion. Taken together, our data show that in Xenopus lung epithelia, the AE, rather than the NKCC, is involved in basolateral Cl− uptake, which contrasts with the common model for Cl− secretion in pulmonary epithelia.

Role of calcium in chloride secretion mediated by cAMP pathway activation in rabbit distal colon mucosa

1993 ◽  
Vol 264 (2) ◽  
pp. G252-G260 ◽  
Author(s):  
V. Calderaro ◽  
E. Chiosi ◽  
R. Greco ◽  
A. M. Spina ◽  
A. Giovane ◽  
...  
Keyword(s):  
Short Circuit ◽  
Fold Increase ◽  
Short Circuit Current ◽  
Distal Colon ◽  
Chloride Secretion ◽  
Free Medium ◽  
Cl Secretion ◽  
Pathway Activation ◽  
Flux Measurements ◽  
Pg E2

Effects of Ca2+ on adenosine 3',5'-cyclic monophosphate (cAMP)-mediated Cl- secretion were investigated in intact mucosa and isolated crypt cells of rabbit descending colon. Addition of 10 microM prostaglandin (PG)E2 or forskolin to tissues incubated in Ca(2+)-free medium increased the size of short-circuit current (Isc) and Cl- secretion as estimated by unidirectional 36Cl flux measurements (net flux = -2.31 +/- 0.24 vs. -1.22 +/- 0.10 mueq.h-1.cm-2, n = 4, P < 0.001). Addition of 10 microM PGE2 to tissues incubated in 1.2 mM Ca2+ Ringer induced a 7-fold increase in mean cAMP level, whereas it produced an 11-fold increase in tissues exposed to Ca(2+)-free medium. Membrane preparations from whole mucosa incubated in Ca(2+)-free medium displayed a cyclic nucleotide phosphodiesterase activity significantly lower than controls (18.76 +/- 0.54 vs. 31.20 +/- 0.39 pmol cAMP. mg protein-1.min-1, means +/- SE, n = 4, P < 0.001). Ca2+ removal also affected adenylate cyclase (AC) responsiveness to agonists; AC activity increased in controls by 54 and 226% after stimulation with 10 microM PGE2 and forskolin, respectively, but it increased more (77 and 325%, respectively) after incubation in Ca(2+)-free solutions.(ABSTRACT TRUNCATED AT 250 WORDS)

Chymotrypsin, ileal chloride transport, and neurotransmitters

1984 ◽  
Vol 247 (3) ◽  
pp. G253-G260 ◽  
Author(s):  
K. A. Hubel
Keyword(s):  
Cell Function ◽  
Chloride Transport ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Electrical Field Stimulation ◽  
Chloride Secretion ◽  
Target Cells ◽  
Flux Chamber ◽  
Rabbit Ileum ◽  
Electrical Field

Electrical field stimulation (EFS) depolarizes nerves and causes chloride secretion by mucosa of rabbit ileum mounted in a flux chamber. To test the hypothesis that the transmitter is a peptide, we determined whether the EFS response was prevented by the endopeptidase chymotrypsin (CT). Serosal, but not mucosal, addition of CT (200 micrograms/ml) reduced the short-circuit current (Isc) response to EFS by 90% or more. CT also reduced Cl absorption by decreasing the mucosal-to-serosal flux, but it did not affect net Na absorption. CT prevented the response to vasoactive intestinal polypeptides, but the response returned when CT activity was eliminated. The response to EFS did not return, however, implying that CT damaged cells that released transmitter or epithelial target cells. CT reduced the Isc response to serotonin by 69% and to A23187 by 10% and did not affect the theophylline response. We conclude that 1) the effects of CT on cell function limit its usefulness in identifying peptide neurotransmitters in epithelium, 2) CT irreversibly inhibits ion transport responses to EFS and to serotonin, and 3) CT reduces absorption of Cl probably by affecting a calcium pathway that modifies Cl transport.

Histamine action on guinea pig ileal mucosa

1984 ◽  
Vol 246 (4) ◽  
pp. G372-G377 ◽  
Author(s):  
H. J. Cooke ◽  
P. R. Nemeth ◽  
J. D. Wood
Keyword(s):  
Receptor Agonist ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Electrical Field Stimulation ◽  
Chloride Secretion ◽  
Evoked Response ◽  
Field Stimulation ◽  
H1 Receptor ◽  
Electrical Field ◽  
Mucosal Response

Nerve-mediated and direct actions of histamine on mucosal transport function in the guinea pig ileum were investigated. Addition of histamine to the serosal side of flat sheet preparations in Ussing chambers evoked a transient increase in base-line short-circuit current that was due primarily to an increase in active chloride secretion. The mucosal response to histamine was mimicked by the H1-receptor agonist 2-methylhistamine, but not by the H2-receptor agonist dimaprit. The histamine-evoked response was prevented by the H1-receptor blocker pyrilamine, but not by the H2-receptor antagonist cimetidine. Thirty percent of the mucosal response to histamine was inhibited by tetrodotoxin. Intracellular electrical recording showed that histamine activated AH/type 2 myenteric neurons, and this response was abolished in the presence of pyrilamine. Local anesthetic action of pyrilamine was ruled out by direct electrical recording from myenteric neurons in the presence and absence of pyrilamine. Electrical field stimulation evoked a biphasic increase in short-circuit current. Histamine and 2-methylhistamine did not alter the sustained phase of the short-circuit current response to electrical field stimulation, although pyrilamine reduced the electrically evoked response by 22%. Muscarinic blockade with atropine reduced the stimulus-evoked response by 55%. When muscarinic receptors were blocked and electrical field stimulation applied, histamine increased the stimulus-evoked mucosal response by 22.3%. These results suggest that histamine increases short-circuit current and chloride secretion by acting at H1-receptor sites on both the enteric innervation of the mucosa and on the enterocytes.

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