Ammonia inhibition of active electrogenic Cl− secretion in both human and rat colon: does a regulatory apical K+ conductance exist?

Butyrate stimulates salt absorption in mammalian colon. We examined whether butyrate also affects Cl- secretion. Mucosal segments of distal colon of male Sprague-Dawley rats and T84 cells were studied in Ussing chambers. In control colon, 1 mM dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP) increased short-circuit current (Isc) and serosal-to-mucosal Cl- flux (JsmCl) by 3.2 +/- 0.8 and 2.9 +/- 0.8 mueq.cm-2.h-1, respectively. Mucosal or serosal 25 mM butyrate prevented DBcAMP-induced increases in Isc and JsmCl. Four and eight millimolar butyrate caused half-maximal inhibition of the increases in JsmCl and Isc, respectively. Butyrate also inhibited basal JsmCl (by 2.0 +/- 0.4 mueq.cm-2.h-1) but not carbachol-mediated Cl- secretion. The relative inhibitory potency at 25 mM of other short-chain fatty acids (SCFA) paralleled their degree of cellular metabolism: butyrate > acetate = propionate > isobutyrate. At 25 mM, all SCFA reduced mucosal intracellular pH (pHi) transiently by 0.1 pH unit. In intact T84 cells, 50 mM butyrate inhibited the DBcAMP-induced rise in Isc by 55%. In T84 cells with nystatin-permeabilized basolateral membranes, butyrate inhibited the increase in Isc by 82%. We conclude that butyrate inhibits basal and cAMP-mediated Cl- secretion by a mechanism independent of pHi, possibly located at the apical membrane.

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Psoralens: novel modulators of Cl- secretion

AJP Cell Physiology ◽

10.1152/ajpcell.1997.272.3.c976 ◽

1997 ◽

Vol 272 (3) ◽

pp. C976-C988 ◽

Cited By ~ 52

Author(s):

D. C. Devor ◽

A. K. Singh ◽

R. J. Bridges ◽

R. A. Frizzell

Keyword(s):

Short Circuit ◽

Basolateral Membrane ◽

Primary Cultures ◽

Receptor Activation ◽

Sustained Response ◽

Rat Colon ◽

Cl Secretion ◽

Cl Channel ◽

Cl Channels ◽

Cl Conductance

We evaluated effects of psoralens on Cl- secretion (short-circuit current, I(sc)) across T84 monolayers. Methoxsalen failed to increase I(sc). Several observations suggest that psoralens open cystic fibrosis transmembrane conductance regulator Cl- channels. 1) After activation of the Ca2+-dependent basolateral membrane K+ channel (K(Ca)) by 1-ethyl-2-benzimidazolinone or thapsigargin, methoxsalen (10 microM) further increased I(sc). 2) When added before carbachol (CCh), methoxsalen potentiated the I(sc) response to CCh, as predicted, if it increased apical Cl- conductance. 3) After establishment of a mucosal-to-serosal Cl- gradient and permeabilization of basolateral membrane with nystatin, psoralens increased Cl- current, which was inhibited by glibenclamide. In contrast, neither TS-TM calix[4]arene nor Cd2+, inhibitors of outwardly rectifying Cl- channels and the ClC-2 Cl-channel, respectively, inhibited psoralen-induced Cl- current. In contrast to their effects on Cl- conductance, psoralens failed to significantly affect basolateral membrane K+ conductance; subsequent addition of 1-ethyl-2-benzimidazolinone induced a large increase in K+ conductance. Also, in excised patches, methoxsalen failed to activate K(Ca). In addition to potentiating the peak response to CCh, psoralens induced a secondary, sustained response. Indeed, when added up to 60 min after return of CCh-induced I(sc) to baseline, psoralens induced a sustained I(sc). This sustained response was inhibited by atropine, demonstrating the requirement for continuous muscarinic receptor activation by CCh. This sustained response was inhibited also by verapamil, removal of bath Ca2+, and charybdotoxin. These results suggest that return of I(sc) to baseline after CCh stimulation is not due to downregulation of Ca2+ influx or K(Ca). Finally, we obtained similar results with psoralens in rat colon and primary cultures of murine tracheal epithelium. On the basis of these observations, we conclude that psoralens represent a novel class of Cl- channel openers that can be used to probe mechanisms underlying Ca2+-mediated Cl- secretion.

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Effects of phorbol esters on sodium and chloride transport in rat colon

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1986.251.4.g509 ◽

1986 ◽

Vol 251 (4) ◽

pp. G509-G517 ◽

Cited By ~ 3

Author(s):

M. Donowitz ◽

H. Y. Cheng ◽

G. W. Sharp

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Transport Systems ◽

Rat Colon ◽

Bathing Solution ◽

Maximal Effect ◽

Dependent Manner ◽

Cl Secretion ◽

Kinase C ◽

Serosal Surface

To determine the role of protein kinase C in the regulation of active electrolyte transport in rat descending colon, the effects of phorbol dibutyrate (PDB) were studied using the Ussing chamber/voltage-clamp technique. PDB added to the serosal surface increased the short-circuit current in a concentration dependent manner with a EC50 of 3 X 10(-8) M and a maximal effect at 10(-7) M PDB. The effect was not seen with the inactive alpha-phorbol analogue but was reproduced with 1-oleoyl-2-acetylglycerol, a more permeable analogue of diacylglycerol. PDB caused a decrease in mucosal-to-serosal and net fluxes of Na and Cl and an increase in serosal-to-mucosal Cl flux, indicating inhibition of Na and Cl absorption and stimulation of Cl secretion. The PDB-induced increase in Cl secretion was virtually abolished by both indomethacin and ibuprofen, indicating a dependence on arachidonic acid metabolism via the cyclooxygenase pathway. The Cl secretion was inhibited by verapamil and Ca2+-free bathing solution on the serosal surface but not by dantrolene, suggesting the importance of extracellular Ca2+ but not intracellular stored Ca2+ in the PDB-induced secretion. The Cl secretory effect was also inhibited by tetrodotoxin and atropine, suggesting involvement of cholinergic nerves. In contrast, the PDB-induced decrease in Na and Cl absorption was not dependent on metabolites of the cyclooxygenase pathway, not dependent on extracellular Ca2+, and not blocked by tetrodotoxin. It appears likely that protein kinase C is involved in the regulation of rat colonic active Na and Cl absorption and electrogenic Cl secretion but that the pathways involved are different in the two transport systems.

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Modulation of secretagogue-induced chloride secretion by intracellular bicarbonate

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1994.266.5.g929 ◽

1994 ◽

Vol 266 (5) ◽

pp. G929-G934 ◽

Cited By ~ 2

Author(s):

P. C. Dagher ◽

T. Z. Morton ◽

C. S. Joo ◽

A. Taglietta-Kohlbrecher ◽

R. W. Egnor ◽

...

Keyword(s):

Cell Line ◽

Short Circuit ◽

Ussing Chamber ◽

Tumor Cell Line ◽

Distal Colon ◽

Rat Colon ◽

Sprague Dawley ◽

T84 Cells ◽

Cl Secretion ◽

Rat Distal Colon

We have previously demonstrated inhibition of basal Cl- secretion by intracellular bicarbonate concentration ([HCO3-]i) in rat distal colon. We now examined whether secretagogue-induced Cl- secretion is inhibited by [HCO3-]i as well. Stripped segments of distal colon from male Sprague-Dawley rats and the colon tumor cell line T84 were studied. Flux measurements were performed in the Ussing chamber under short-circuit conditions. [HCO3-]i was calculated from intracellular pH (pHi) values that were estimated with the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. Dibutyryl adenosine 3',5'-cyclic monophosphate (cAMP) and carbachol were used as secretagogues. In both distal colon and T84 cells, [HCO3-]i did not affect cAMP-induced Cl- secretion. However, carbachol-induced secretion was inhibited by [HCO3-]i; in rat colon, Cl- secretion decreased from 2.3 to 1.5 mueq.cm-2.h-1 when [HCO3-]i was increased from 15.0 to 28.4 mM (P < 0.05). In T84 cells, the change in short-circuit current decreased from 8.1 to 1.1 microA/cm2 over a range of [HCO3-]i from 0 to 15.6 mM (P < 0.001). We conclude that [HCO3-]i is an important modulator of carbachol-stimulated Cl- secretion in both rat distal colon and the T84 cell line. cAMP-mediated secretion is not affected by [HCO3-]i.

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Thromboxane A2, released by the anti- tumour drug irinotecan, is a novel stimulator of Cl−secretion in isolated rat colon

The Journal of Physiology ◽

10.1111/j.1469-7793.1997.133bc.x ◽

1997 ◽

Vol 505 (1) ◽

pp. 133-144 ◽

Cited By ~ 32

Author(s):

Hideki Sakai ◽

Takahiro Sato ◽

Noriko Hamada ◽

Miyuki Yasue ◽

Akira Ikari ◽

...

Keyword(s):

Thromboxane A2 ◽

Rat Colon ◽

Cl Secretion ◽

Isolated Rat ◽

Isolated Rat Colon

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Forskolin- but not ionomycin-evoked Cl- secretion in colonic epithelia depends on intact microtubules

AJP Cell Physiology ◽

10.1152/ajpcell.1994.266.3.c661 ◽

1994 ◽

Vol 266 (3) ◽

pp. C661-C668 ◽

Cited By ~ 27

Author(s):

C. M. Fuller ◽

R. J. Bridges ◽

D. J. Benos

Keyword(s):

Short Circuit ◽

Physiological Role ◽

Short Circuit Current ◽

Inhibitory Effect ◽

Secretory Response ◽

Rat Colon ◽

Epithelial Cell Line ◽

Cl Secretion ◽

Stimulus Response ◽

Cl Channels

Several transport proteins are known to be trafficked to the cell membrane in response to appropriate secretagogues. In several cases, the response has been shown to be dependent on the cytoskeleton. We tested the hypothesis that the forskolin- and/or ionomycin-sensitive Cl- secretory response in colonic epithelia is dependent on an intact cytoskeleton. Using 125I- efflux as an assay for Cl- transport in the colonic epithelial cell line T84, we found that preincubation of the tissue for 3 h with either of two inhibitors of microtubule polymerization, nocodazole or colchicine, disrupted the cellular tubulin architecture and also reduced the forskolin- but not the ionomycin-evoked I- efflux. In contrast, brief exposure (4 min) to nocodazole was without effect on the forskolin-sensitive efflux, suggesting that the drug is not acting to block the stimulus-response pathway. An inactive structural analogue of colchicine, beta-lumicolchicine, had no inhibitory effect on either the forskolin-sensitive efflux or on microtubular structure. In a second model of Cl- secretion, the stripped rat colon, both colchicine and nocodazole reduced the forskolin-dependent short-circuit current by an average of 30-40%, suggesting a similar mechanism for insertion of Cl- channels into the plasma membrane. These findings suggest that the Cl- secretory response is dependent on microtubules and has a physiological role in the adenosine 3',5'-cyclic monophosphate-dependent, but not the Ca(2+)-dependent, Cl- secretion in colonic epithelia.

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