Ammonia effect on calcium-activated chloride secretion in T84 intestinal epithelial monolayers

1997 ◽  
Vol 273 (2) ◽  
pp. C634-C642 ◽  
Author(s):  
J. M. Mayol ◽  
B. J. Hrnjez ◽  
H. I. Akbarali ◽  
J. C. Song ◽  
J. A. Smith ◽  
...  
Keyword(s):  
Inhibitory Action ◽  
Chloride Secretion ◽  
Secretory Response ◽  
Transepithelial Transport ◽  
K Channel ◽  
Cl Secretion ◽  
Fura 2 ◽  
Intestinal Epithelia ◽  
Efflux Rate Constant ◽  
High Concentrations

We recently showed that ammonia profoundly inhibits cyclic nucleotide-regulated Cl- secretion in model human T84 intestinal epithelia but does not impair the secretory response to the Ca2+ agonist carbachol. Using transepithelial transport, fura 2 fluorescence, and radioisotopic efflux techniques, we further explored this dichotomy and arrived at a preliminary explanation for the inhibitory action of ammonia. The secretory response to the Ca(2+)-adenosinetriphosphatase inhibitor thapsigargin is unaffected by ammonia, which suggests that an increase in intracellular Ca2+ stimulates secretory pathways that are insensitive to ammonia. Surprisingly, Cl- secretion elicited by the Ca2+ ionophores ionomycin and A23187 is markedly blunted in monolayers pretreated with ammonia. However, ammonia posttreatment does not inhibit the secretory response to ionophore, which suggests that ammonia may interfere with the ability of these ionophores to increase intracellular [Ca2+]. This hypothesis is directly supported by fura 2 experiments. The inhibitory action of ammonia parallels the behavior of the K+ channel blocker Ba2+, and ammonia reduces the basolateral 86Rb+ efflux rate constant in forskolin- but not in carbachol-treated monolayers. Ammonia, which is present in high concentrations in the normal gastro-intestinal tract, may serve as a novel endogenous regulator of epithelial electrolyte transport by interfering with a Ba(2+)-sensitive basolateral K+ conductance distinct from the Ca(2+)-activated basolateral K+ conductance.

Inhibition of ATP levels by quinidine in a human colonic epithelial cell line

1986 ◽  
Vol 250 (6) ◽  
pp. G806-G813
Author(s):  
K. Dharmsathaphorn ◽  
P. Huott ◽  
C. A. Cartwright ◽  
J. A. McRoberts ◽  
K. G. Mandel ◽  
...  
Keyword(s):  
Inhibitory Action ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Human Colon ◽  
Inhibitory Effect ◽  
K Channel ◽  
Epithelial Cell Line ◽  
Cl Secretion ◽  
Atp Levels ◽  
Similar Inhibitory Effect

The influence of quinidine, a putative K+ channel blocker, on Cl- secretion induced by vasoactive intestinal polypeptide (VIP) was investigated. Quinidine inhibited Cl- secretion induced by VIP in T84 cell monolayers. A similar inhibitory effect of quinidine on Cl- secretion was observed in an isolated human colon. However, in the isolated human colon, which absorbs Na+ avidly, inhibition of Na+ absorption predominated. In the T84 cell, the half-maximal inhibition by quinidine occurred at 60 microM, while 300 microM almost completely inhibited the VIP-induced Cl— secretion. Mucosal addition of quinidine was at least equally effective compared with serosal addition, suggesting that quinidine acts on the apical membrane or intracellularly. Quinidine had little or no effect on VIP-stimulated Cl- efflux in the first 15 min after its addition, suggesting that blockage of the Cl- exit pathway on the apical membrane is an unlikely mechanism. Similarly, quinidine did not inhibit the K+-recycling mechanism on the basolateral membrane in the first 15 min after its addition. The initial inhibitory action of quinidine corresponded better with its ability to decrease cellular ATP levels. Our study suggests that the depletion of cellular ATP levels may explain the initial inhibitory action of quinidine on electrolyte transport in the intestine, while the late effect is multifactorial.

Modulation of Cl- secretion by benzimidazolones. II. Coordinate regulation of apical GCl and basolateral GK

1996 ◽  
Vol 271 (5) ◽  
pp. L785-L795 ◽  
Author(s):  
D. C. Devor ◽  
A. K. Singh ◽  
R. J. Bridges ◽  
R. A. Frizzell
Keyword(s):  
Cystic Fibrosis ◽  
Basolateral Membrane ◽  
Primary Cultures ◽  
Secretory Response ◽  
K Channel ◽  
T84 Cells ◽  
Coordinate Regulation ◽  
Cl Secretion ◽  
Cl Channels ◽  
Subsequent Addition

We previously demonstrated that the novel benzimidazolone, 1-ethyl-2-benzimidazolinone (1-EBIO), stimulates a sustained Cl- secretory response across T84 monolayers by opening a Ca(2+)-dependent basolateral K+ channel. In the present work, we evaluated the effects on Cl-secretion of other benzimidazolones, NS-004 and NS-1619, which have been shown to open cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels. In contrast to 1-EBIO, neither NS-004 nor NS-1619 stimulated a significant Cl- secretory current (Isc). Neither NS-004 nor NS-1619 increased Isc subsequent to forskolin stimulation. However, when added after 1-EBIO, NS-004 and NS-1619 stimulated large sustained increases in Isc. In addition, NS-004 and NS-1619 potentiated the effects of carbachol. We used nystatin to permeabilize the apical or basolateral membrane to determine the effects of NS-004 and 1-EBIO on the basolateral K+ (IK) and apical Cl- (ICl) currents. Both NS-004 and 1-EBIO increased ICl, and the stimulated currents were inhibited by glibenclamide. In contrast, NS-004 failed to significantly affect IK, but subsequent addition of 1-EBIO induced a large increase in IK. The effects of 1-EBIO, NS-004, and NS-1619 on the Ca(2+)-dependent K+ channel (KCa) in T84 cells was determined in excised inside-out patches. Neither NS-004 nor NS-1619 affected K+ channel activity, whereas the subsequent addition of 1-EBIO produced a marked channel activation. Results similar to those observed in T84 monolayers were obtained from murine airway cell primary cultures: NS-004 or NS-1619 had no effect on Isc, whereas 1-EBIO stimulated a sustained Cl- secretory response. The results demonstrate that activation of CFTR alone is insufficient to evoke transepithelial Cl- secretion. Activation of the basolateral membrane K+ channel is a necessary component of the secretory response. Thus the basolateral membrane KCa may be a novel pharmacological target in cystic fibrosis therapy.

Modulation of Cl- secretion by benzimidazolones. I. Direct activation of a Ca(2+)-dependent K+ channel

1996 ◽  
Vol 271 (5) ◽  
pp. L775-L784 ◽  
Author(s):  
D. C. Devor ◽  
A. K. Singh ◽  
R. A. Frizzell ◽  
R. J. Bridges
Keyword(s):  
Short Circuit ◽  
Basolateral Membrane ◽  
Primary Cultures ◽  
Short Circuit Current ◽  
Secretory Response ◽  
K Channel ◽  
T84 Cells ◽  
K Channels ◽  
Cl Secretion ◽  
Direct Activation

We evaluated the effects of the novel benzimidazolone, 1-ethyl-2-benzimidazolinone (1-EBIO), on Cl- secretion across T84 monolayers. 1-EBIO stimulated a sustained Cl- secretory response at a half-maximal effective concentration of 490 microM. Charybdotoxin (CTX) inhibited the 1-EBIO-induced short-circuit current (Isc) with an inhibitory constant (Ki) of 3.6 nM, whereas 293B, an inhibitor of adenosine 3',5'-cyclic monophosphate-activated K+ channels, had no effect on the current induced by 1-EBIO. In contrast, CTX failed to inhibit the 293B-sensitive forskolin-induced Isc. The above results suggested that 1-EBIO may be activating the basolateral membrane Ca(2+)-dependent K+ channel (KCa) in these cells. This was further confirmed using nystatin to permeabilize the apical membrane in the presence of a mucosa-to-serosa K+ gradient and determining the effects of 1-EBIO on the basolateral K+ current (IK). Under these conditions, 1-EBIO induced a large increase in IK that was blocked by CTX. In membrane vesicles prepared from T84 cells, 1-EBIO stimulated 86Rb+ uptake in a CTX-sensitive manner; the Ki for inhibition by CTX was 3.5 nM. Similar to our intact monolayer studies, this 86Rb+ uptake was not blocked by 293B. The effects of 1-EBIO on the KCa in T84 cells was determined in excised inside-out patches. 1-EBIO (100 microM) increased the product of the number of channels and the open channel probability from 0.09 +/- 0.03 to 1.17 +/- 0.27 (n = 8); this effect on KCa activity required a minimal level of free Ca2+. Similar to its effect on T84 cells, 1-EBIO stimulated a sustained Cl- secretory current in rat colonic epithelium, which was partially blocked by CTX. Finally, 1-EBIO stimulated a sustained Cl- secretory response in primary cultures of murine tracheal epithelium. We conclude that the benzimidazolone, 1-EBIO, stimulates Cl- secretion in secretory epithelia via the direct activation of a Kca. 1-EBIO is the first pharmacological opener of this important class of epithelial K+ channels to be identified.

Duramycin enhances chloride secretion in airway epithelium

1990 ◽  
Vol 259 (3) ◽  
pp. C450-C454 ◽  
Author(s):  
M. M. Cloutier ◽  
L. Guernsey ◽  
P. Mattes ◽  
B. Koeppen
Keyword(s):  
Airway Epithelium ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Chloride Secretion ◽  
Bathing Solution ◽  
Maximum Increase ◽  
Ussing Chambers ◽  
Cl Secretion ◽  
Polypeptide Antibiotic ◽  
High Concentrations

The effect of duramycin, a polypeptide antibiotic, on Cl- transport in canine tracheal epithelium mounted in Ussing chambers was studied. Over a narrow concentration range, duramycin increased short-circuit current (Isc) and net Cl- secretion and had no effect on mannitol flux when added to the mucosal bathing solution. The maximum increase in Isc was observed at a duramycin concentration of 2 X 10(-6) M and was associated with an increase in both unidirectional Cl- fluxes. Higher duramycin concentrations produced a decrease in Isc. Submucosal addition of duramycin had no effect on Isc except at high concentrations. Pretreatment of tissues with mucosal amiloride (10(-4) M) to reduce basal Na+ transport had no effect on the subsequent response to duamycin. In other tissues pretreated with 10(-3) M dibutyryl adenosine 3',5'-cyclic monophosphate (cAMP), duramycin produced a further increase in Isc and net Cl- secretion similar to its effect in nonpretreated tissues. In all instances the increase in Isc was entirely accounted for by an increase in net Cl- secretion. We conclude that duramycin increases Isc and Cl- secretion in airway epithelium. Although the mechanism of activation is not known, these data demonstrate that duramycin increases Cl- secretion by a pathway other than cAMP. An understanding of the mechanism of action of duramycin may further our understanding of Cl- secretion regulation in airway epithelium.

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.

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.

THE SELECTIVE PROTEOLYTIC ENZYME INHIBITORY ACTION OF BENZETHONIUM CHLORIDE

1960 ◽  
Vol 38 (1) ◽  
pp. 25-32 ◽  
Author(s):  
Ivan T. Beck ◽  
E. Pinter ◽  
R. D. McKenna ◽  
H. Griff
Keyword(s):  
Subcutaneous Injection ◽  
Proteolytic Enzyme ◽  
Inhibitory Action ◽  
Hemorrhagic Pancreatitis ◽  
Benzethonium Chloride ◽  
Tryptic Activity ◽  
High Concentrations ◽  
Acute Hemorrhagic Pancreatitis ◽  
Very High

Acute hemorrhagic pancreatitis in humans is thought to be perpetuated by the autolytic processes catalyzed by trypsin and lipase. This study is an integral part of our search for trypsin and lipase inhibitors to be used in the treatment of this disease.Benzethonium chloride was found to inhibit tryptic activity in vitro. The proteolytic activity of rabbit's serum was inhibited, and the inhibition was most pronounced 6 to 12 hours after the subcutaneous injection of the compound. Fibrinolysin was also inhibited in vitro but benzethonium chloride had no inhibitory action on chymotrypsin, pepsin, or lipase.Serum proteins in vitro were precipitated only with very high concentrations of the compound. No significant protein changes were observed in sera of rabbits after the subcutaneous injection of the compound.

scholarly journals Zinc inhibits cAMP-stimulated Cl secretion via basolateral K-channel blockade in rat ileum

2005 ◽  
Vol 288 (5) ◽  
pp. G956-G963 ◽  
Author(s):  
Kazi Mirajul Hoque ◽  
Vazhaikkurichi M. Rajendran ◽  
Henry J. Binder
Keyword(s):  
Short Circuit ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
K Channel ◽  
Isolated Cells ◽  
Rat Ileum ◽  
Anion Secretion ◽  
Cl Secretion ◽  
Ion Absorption ◽  
Isotonic Buffer

Zn, an essential micronutrient and second most abundant trace element in cell and tissues, reduces stool output when administered to children with acute diarrhea. The mechanism by which Zn improves diarrhea is not known but could result from stimulating Na absorption and/or inhibiting anion secretion. The aim of this study was to investigate the direct effect of Zn on intestinal epithelial ion absorption and secretion. Rat ileum was partially stripped of serosal and muscle layers, and the mucosa was mounted in lucite chambers. Potential difference and short-circuit current were measured by conventional current-voltage clamp method.86Rb efflux and uptake were assessed for serosal K channel and Na-K-2Cl cotransport activity, respectively. Efflux experiments were performed in isolated cells preloaded with86Rb in the presence of ouabain and bumetanide, whereas uptake experiments were performed in low-Cl isotonic buffer containing Ba and ouabain. Neither mucosal nor serosal Zn affected glucose-stimulated Na absorption. In contrast, forskolin-induced Cl secretion was markedly reduced by serosal but not mucosal addition of Zn. Zn also substantially reversed the increase in Cl secretion induced by 8-bromoadenosine 3′,5′-cyclic monophosphate (8-BrcAMP) with half-maximal inhibitory concentration of 0.43 mM. In contrast, serosal Zn did not alter Cl secretion stimulated by carbachol, a Ca-dependent agonist. Zn inhibited 8-BrcAMP-stimulated86Rb efflux but not carbachol-stimulated86Rb efflux. Zn had no effect on bumetanide-sensitive86Rb uptake, Na-K-ATPase, or CFTR. We conclude from these studies that Zn inhibits cAMP-induced Cl secretion by blocking basolateral membrane K channels.

scholarly journals Carbon Monoxide Inhibits Cytokine and Chloride Secretion in Human Bronchial Epithelia

2018 ◽  
Vol 49 (2) ◽  
pp. 626-637 ◽  
Author(s):  
Rui-Gang Zhang ◽  
Chung-Yin Yip ◽  
Wing-hung Ko
Keyword(s):  
Carbon Monoxide ◽  
Ion Transport ◽  
Cell Damage ◽  
Chloride Secretion ◽  
Bronchial Epithelial Cell ◽  
Purinergic Signalling ◽  
Epithelial Cell Line ◽  
Cl Secretion ◽  
Bronchial Epithelia ◽  
Anti Inflammatory

Background/Aims: Carbon monoxide (CO) is an important gas produced endogenously by heme oxygenase (HO) that functions as an anti-inflammatory and in ion channel modulation, but the effects of CO on airway inflammation and ion transport remains unclear. Methods: The effect of CO on cell damage- and nucleotide-induced pro-inflammatory cytokine release in primary human bronchial epithelia cells (HBE) and in the 16HBE14o- human bronchial epithelial cell line were investigated. The effects of CO on calcium- and cAMP-dependent chloride (Cl-) secretion were examined using a technique that allowed the simultaneous measurement and quantification of real-time changes in signalling molecules (cAMP and Ca2+) and ion transport in a polarised epithelium. Results: CO suppressed the release of interleukin (IL)-6 and IL-8 and decreased the phosphorylation of ERK1/2 and NF-κB p65. Furthermore, CO inhibited UTP-induced increases in calcium and Cl- secretion, and forskolin-induced increases in cAMP and Cl- secretion. Conclusions: These findings suggest a novel anti-inflammatory role of CO in human bronchial epithelia via interactions with purinergic signalling pathways. Further, CO modulated both the Ca2+- and cAMP-dependent secretion of Cl-.

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)

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