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. 1997 Dec 15;100(12):3111–3120. doi: 10.1172/JCI119866

The antifungal antibiotic, clotrimazole, inhibits chloride secretion by human intestinal T84 cells via blockade of distinct basolateral K+ conductances. Demonstration of efficacy in intact rabbit colon and in an in vivo mouse model of cholera.

P A Rufo 1, D Merlin 1, M Riegler 1, M H Ferguson-Maltzman 1, B L Dickinson 1, C Brugnara 1, S L Alper 1, W I Lencer 1
PMCID: PMC508524  PMID: 9399958

Abstract

The antifungal antibiotic clotrimazole (CLT) blocks directly and with high potency the Ca2+-activated K+ channels of human erythrocytes, erythroleukemia cells, and ferret vascular smooth muscle cells. We recently reported that CLT inhibits Cl- secretion in human intestinal T84 cells, likely by affecting K+ transport (Rufo, P.A., L. Jiang, S.J. Moe, C. Brugnara, S.L. Alper, and W.I. Lencer. 1996. J. Clin. Invest. 98:2066-2075). To determine if CLT had direct effects on K+ conductances in T84 cells, we selectively permeabilized apical membranes of confluent T84 cell monolayers using the ionophore amphotericin B. This technique permits direct measurement of basolateral K+ transport. We found that CLT and a stable des-imidazolyl derivative inhibited directly two pharmacologically distinct basolateral membrane K+conductances, but had no effect on apical membrane Cl- conductances. The effects of CLT on Cl- secretion were also examined in intact tissue. CLT inhibited forskolin-induced Cl- secretion in rabbit colonic mucosal sheets mounted in Ussing chambers by 91%. CLT also inhibited cholera toxin-induced intestinal Cl- secretion in intact mice by 94%. These data provide direct evidence that CLT blocks Cl- secretion in intestinal T84 cells by inhibition of basolateral K+ conductances, and show that CLT inhibits salt and water secretion from intact tissue in vitro and in vivo. The results further support the suggestion that CLT and its metabolites may show clinical efficacy in the treatment of secretory diarrheas of diverse etiologies.

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Selected References

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  1. Bachmann K. A., Madhira M. S., Rankin G. O. The effects of cobalt chloride, SKF-525A, and N-(3,5-dichlorophenyl)succinimide on in vivo hepatic mixed function oxidase activity as determined by single-sample plasma clearances. Xenobiotica. 1992 Jan;22(1):27–31. doi: 10.3109/00498259209053099. [DOI] [PubMed] [Google Scholar]
  2. Barrett K. E. Positive and negative regulation of chloride secretion in T84 cells. Am J Physiol. 1993 Oct;265(4 Pt 1):C859–C868. doi: 10.1152/ajpcell.1993.265.4.C859. [DOI] [PubMed] [Google Scholar]
  3. Bleich M., Riedemann N., Warth R., Kerstan D., Leipziger J., Hör M., Driessche W. V., Greger R. Ca2+ regulated K+ and non-selective cation channels in the basolateral membrane of rat colonic crypt base cells. Pflugers Arch. 1996 Oct;432(6):1011–1022. doi: 10.1007/s004240050229. [DOI] [PubMed] [Google Scholar]
  4. Brugnara C., Armsby C. C., Sakamoto M., Rifai N., Alper S. L., Platt O. Oral administration of clotrimazole and blockade of human erythrocyte Ca(++)-activated K+ channel: the imidazole ring is not required for inhibitory activity. J Pharmacol Exp Ther. 1995 Apr;273(1):266–272. [PubMed] [Google Scholar]
  5. Brugnara C., De Franceschi L., Alper S. L. Ca(2+)-activated K+ transport in erythrocytes. Comparison of binding and transport inhibition by scorpion toxins. J Biol Chem. 1993 Apr 25;268(12):8760–8768. [PubMed] [Google Scholar]
  6. Brugnara C., Gee B., Armsby C. C., Kurth S., Sakamoto M., Rifai N., Alper S. L., Platt O. S. Therapy with oral clotrimazole induces inhibition of the Gardos channel and reduction of erythrocyte dehydration in patients with sickle cell disease. J Clin Invest. 1996 Mar 1;97(5):1227–1234. doi: 10.1172/JCI118537. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Brugnara C., de Franceschi L., Alper S. L. Inhibition of Ca(2+)-dependent K+ transport and cell dehydration in sickle erythrocytes by clotrimazole and other imidazole derivatives. J Clin Invest. 1993 Jul;92(1):520–526. doi: 10.1172/JCI116597. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Capdevila J., Gil L., Orellana M., Marnett L. J., Mason J. I., Yadagiri P., Falck J. R. Inhibitors of cytochrome P-450-dependent arachidonic acid metabolism. Arch Biochem Biophys. 1988 Mar;261(2):257–263. doi: 10.1016/0003-9861(88)90340-2. [DOI] [PubMed] [Google Scholar]
  9. Cartwright C. A., McRoberts J. A., Mandel K. G., Dharmsathaphorn K. Synergistic action of cyclic adenosine monophosphate- and calcium-mediated chloride secretion in a colonic epithelial cell line. J Clin Invest. 1985 Nov;76(5):1837–1842. doi: 10.1172/JCI112176. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. De Franceschi L., Saadane N., Trudel M., Alper S. L., Brugnara C., Beuzard Y. Treatment with oral clotrimazole blocks Ca(2+)-activated K+ transport and reverses erythrocyte dehydration in transgenic SAD mice. A model for therapy of sickle cell disease. J Clin Invest. 1994 Apr;93(4):1670–1676. doi: 10.1172/JCI117149. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Devor D. C., Duffey M. E. Carbachol induces K+, Cl-, and nonselective cation conductances in T84 cells: a perforated patch-clamp study. Am J Physiol. 1992 Oct;263(4 Pt 1):C780–C787. doi: 10.1152/ajpcell.1992.263.4.C780. [DOI] [PubMed] [Google Scholar]
  12. Devor D. C., Frizzell R. A. Calcium-mediated agonists activate an inwardly rectified K+ channel in colonic secretory cells. Am J Physiol. 1993 Nov;265(5 Pt 1):C1271–C1280. doi: 10.1152/ajpcell.1993.265.5.C1271. [DOI] [PubMed] [Google Scholar]
  13. Devor D. C., Simasko S. M., Duffey M. E. Carbachol induces oscillations of membrane potassium conductance in a colonic cell line, T84. Am J Physiol. 1990 Feb;258(2 Pt 1):C318–C326. doi: 10.1152/ajpcell.1990.258.2.C318. [DOI] [PubMed] [Google Scholar]
  14. Devor D. C., Singh A. K., Bridges R. J., Frizzell R. A. Modulation of Cl- secretion by benzimidazolones. II. Coordinate regulation of apical GCl and basolateral GK. Am J Physiol. 1996 Nov;271(5 Pt 1):L785–L795. doi: 10.1152/ajplung.1996.271.5.L785. [DOI] [PubMed] [Google Scholar]
  15. Dharmsathaphorn K., Madara J. L. Established intestinal cell lines as model systems for electrolyte transport studies. Methods Enzymol. 1990;192:354–389. doi: 10.1016/0076-6879(90)92082-o. [DOI] [PubMed] [Google Scholar]
  16. Dharmsathaphorn K., Pandol S. J. Mechanism of chloride secretion induced by carbachol in a colonic epithelial cell line. J Clin Invest. 1986 Feb;77(2):348–354. doi: 10.1172/JCI112311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Duffey M. E., Devor D. C., Ahmed Z., Simasko S. M. Characterization of a membrane potassium ion conductance in intestinal secretory cells using whole cell patch-clamp and calcium ion-sensitive dye techniques. Methods Enzymol. 1990;192:309–324. doi: 10.1016/0076-6879(90)92079-s. [DOI] [PubMed] [Google Scholar]
  18. Feil W., Lacy E. R., Wong Y. M., Burger D., Wenzl E., Starlinger M., Schiessel R. Rapid epithelial restitution of human and rabbit colonic mucosa. Gastroenterology. 1989 Sep;97(3):685–701. doi: 10.1016/0016-5085(89)90640-9. [DOI] [PubMed] [Google Scholar]
  19. Fischer H., Kreusel K. M., Illek B., Machen T. E., Hegel U., Clauss W. The outwardly rectifying Cl- channel is not involved in cAMP-mediated Cl- secretion in HT-29 cells: evidence for a very-low-conductance Cl- channel. Pflugers Arch. 1992 Nov;422(2):159–167. doi: 10.1007/BF00370415. [DOI] [PubMed] [Google Scholar]
  20. Ginsburg C. H., Braden G. L., Tauber A. I., Trier J. S. Oral clotrimazole in the treatment of esophageal candidiasis. Am J Med. 1981 Nov;71(5):891–895. doi: 10.1016/0002-9343(81)90393-4. [DOI] [PubMed] [Google Scholar]
  21. Grass G. M., Sweetana S. A. In vitro measurement of gastrointestinal tissue permeability using a new diffusion cell. Pharm Res. 1988 Jun;5(6):372–376. doi: 10.1023/a:1015911712079. [DOI] [PubMed] [Google Scholar]
  22. Halm D. R., Rechkemmer G. R., Schoumacher R. A., Frizzell R. A. Apical membrane chloride channels in a colonic cell line activated by secretory agonists. Am J Physiol. 1988 Apr;254(4 Pt 1):C505–C511. doi: 10.1152/ajpcell.1988.254.4.C505. [DOI] [PubMed] [Google Scholar]
  23. Huflejt M. E., Blum R. A., Miller S. G., Moore H. P., Machen T. E. Regulated Cl transport, K and Cl permeability, and exocytosis in T84 cells. J Clin Invest. 1994 May;93(5):1900–1910. doi: 10.1172/JCI117181. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Iliev I. G., Marino A. A. Potassium channels in epithelial cells. Cell Mol Biol Res. 1993;39(6):601–611. [PubMed] [Google Scholar]
  25. Ipp M. M., Boxall L., Gelfand E. W. Clotrimazole: intermittent therapy in chronic mucocutaneous candidiasis. Am J Dis Child. 1977 Mar;131(3):305–307. doi: 10.1001/archpedi.1977.02120160059009. [DOI] [PubMed] [Google Scholar]
  26. Kirk K. L., Dawson D. C. Basolateral potassium channel in turtle colon. Evidence for single-file ion flow. J Gen Physiol. 1983 Sep;82(3):297–313. doi: 10.1085/jgp.82.3.297. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kitazono T., Heistad D. D., Faraci F. M. Dilatation of the basilar artery in response to selective activation of endothelin B receptors in vivo. J Pharmacol Exp Ther. 1995 Apr;273(1):1–6. [PubMed] [Google Scholar]
  28. Leikin S., Parrott R., Randolph J. Clotrimazole treatment of chronic mucocutaneous candidiasis. J Pediatr. 1976 May;88(5):864–866. doi: 10.1016/s0022-3476(76)81133-x. [DOI] [PubMed] [Google Scholar]
  29. Lencer W. I., Delp C., Neutra M. R., Madara J. L. Mechanism of cholera toxin action on a polarized human intestinal epithelial cell line: role of vesicular traffic. J Cell Biol. 1992 Jun;117(6):1197–1209. doi: 10.1083/jcb.117.6.1197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Lencer W. I., de Almeida J. B., Moe S., Stow J. L., Ausiello D. A., Madara J. L. Entry of cholera toxin into polarized human intestinal epithelial cells. Identification of an early brefeldin A sensitive event required for A1-peptide generation. J Clin Invest. 1993 Dec;92(6):2941–2951. doi: 10.1172/JCI116917. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Lohrmann E., Burhoff I., Nitschke R. B., Lang H. J., Mania D., Englert H. C., Hropot M., Warth R., Rohm W., Bleich M. A new class of inhibitors of cAMP-mediated Cl- secretion in rabbit colon, acting by the reduction of cAMP-activated K+ conductance. Pflugers Arch. 1995 Feb;429(4):517–530. doi: 10.1007/BF00704157. [DOI] [PubMed] [Google Scholar]
  32. Lomax R. B., Warhurst G., Sandle G. I. Characteristics of two basolateral potassium channel populations in human colonic crypts. Gut. 1996 Feb;38(2):243–247. doi: 10.1136/gut.38.2.243. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. MacVinish L. J., Pickles R. J., Cuthbert A. W. Cyclic AMP and Ca2+ interactions affecting epithelial chloride secretion in human cultured colonic epithelia. Br J Pharmacol. 1993 Feb;108(2):462–468. doi: 10.1111/j.1476-5381.1993.tb12826.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Madara J. L., Parkos C., Colgan S., MacLeod R. J., Nash S., Matthews J., Delp C., Lencer W. Cl- secretion in a model intestinal epithelium induced by a neutrophil-derived secretagogue. J Clin Invest. 1992 Jun;89(6):1938–1944. doi: 10.1172/JCI115800. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Madara J. L., Stafford J., Dharmsathaphorn K., Carlson S. Structural analysis of a human intestinal epithelial cell line. Gastroenterology. 1987 May;92(5 Pt 1):1133–1145. doi: 10.1016/s0016-5085(87)91069-9. [DOI] [PubMed] [Google Scholar]
  36. Mandel K. G., McRoberts J. A., Beuerlein G., Foster E. S., Dharmsathaphorn K. Ba2+ inhibition of VIP- and A23187-stimulated Cl- secretion by T84 cell monolayers. Am J Physiol. 1986 Mar;250(3 Pt 1):C486–C494. doi: 10.1152/ajpcell.1986.250.3.C486. [DOI] [PubMed] [Google Scholar]
  37. Merlin D., Guo X., Laboisse C. L., Hopfer U. Ca2+ and cAMP activate different K+ conductances in the human intestinal goblet cell line HT29-Cl.16E. Am J Physiol. 1995 Jun;268(6 Pt 1):C1503–C1511. doi: 10.1152/ajpcell.1995.268.6.C1503. [DOI] [PubMed] [Google Scholar]
  38. Powell D. W. Barrier function of epithelia. Am J Physiol. 1981 Oct;241(4):G275–G288. doi: 10.1152/ajpgi.1981.241.4.G275. [DOI] [PubMed] [Google Scholar]
  39. Reenstra W. W. Inhibition of cAMP- and Ca-dependent Cl- secretion by phorbol esters: inhibition of basolateral K+ channels. Am J Physiol. 1993 Jan;264(1 Pt 1):C161–C168. doi: 10.1152/ajpcell.1993.264.1.C161. [DOI] [PubMed] [Google Scholar]
  40. Riegler M., Sedivy R., Pothoulakis C., Hamilton G., Zacherl J., Bischof G., Cosentini E., Feil W., Schiessel R., LaMont J. T. Clostridium difficile toxin B is more potent than toxin A in damaging human colonic epithelium in vitro. J Clin Invest. 1995 May;95(5):2004–2011. doi: 10.1172/JCI117885. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Riegler M., Sedivy R., Sogukoglu T., Cosentini E., Bischof G., Teleky B., Feil W., Schiessel R., Hamilton G., Wenzl E. Epidermal growth factor promotes rapid response to epithelial injury in rabbit duodenum in vitro. Gastroenterology. 1996 Jul;111(1):28–36. doi: 10.1053/gast.1996.v111.pm8698221. [DOI] [PubMed] [Google Scholar]
  42. Rittenhouse A. R., Parker C., Brugnara C., Morgan K. G., Alper S. L. Inhibition of maxi-K currents in ferret portal vein smooth muscle cells by the antifungal clotrimazole. Am J Physiol. 1997 Jul;273(1 Pt 1):C45–C56. doi: 10.1152/ajpcell.1997.273.1.C45. [DOI] [PubMed] [Google Scholar]
  43. Rufo P. A., Jiang L., Moe S. J., Brugnara C., Alper S. L., Lencer W. I. The antifungal antibiotic, clotrimazole, inhibits Cl- secretion by polarized monolayers of human colonic epithelial cells. J Clin Invest. 1996 Nov 1;98(9):2066–2075. doi: 10.1172/JCI119012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Sawyer P. R., Brogden R. N., Pinder R. M., Speight T. M., Avery Clotrimazole: a review of its antifungal activity and therapeutic efficacy. Drugs. 1975;9(6):424–447. doi: 10.2165/00003495-197509060-00003. [DOI] [PubMed] [Google Scholar]
  45. Sears C. L., Firoozmand F., Mellander A., Chambers F. G., Eromar I. G., Bot A. G., Scholte B., De Jonge H. R., Donowitz M. Genistein and tyrphostin 47 stimulate CFTR-mediated Cl- secretion in T84 cell monolayers. Am J Physiol. 1995 Dec;269(6 Pt 1):G874–G882. doi: 10.1152/ajpgi.1995.269.6.G874. [DOI] [PubMed] [Google Scholar]
  46. Tabcharani J. A., Boucher A., Eng J. W., Hanrahan J. W. Regulation of an inwardly rectifying K channel in the T84 epithelial cell line by calcium, nucleotides and kinases. J Membr Biol. 1994 Nov;142(2):255–266. doi: 10.1007/BF00234947. [DOI] [PubMed] [Google Scholar]
  47. Tousson A., Fuller C. M., Benos D. J. Apical recruitment of CFTR in T-84 cells is dependent on cAMP and microtubules but not Ca2+ or microfilaments. J Cell Sci. 1996 Jun;109(Pt 6):1325–1334. doi: 10.1242/jcs.109.6.1325. [DOI] [PubMed] [Google Scholar]
  48. Wang W. H., Lu M., Hebert S. C. Cytochrome P-450 metabolites mediate extracellular Ca(2+)-induced inhibition of apical K+ channels in the TAL. Am J Physiol. 1996 Jul;271(1 Pt 1):C103–C111. doi: 10.1152/ajpcell.1996.271.1.C103. [DOI] [PubMed] [Google Scholar]
  49. Warth R., Riedemann N., Bleich M., Van Driessche W., Busch A. E., Greger R. The cAMP-regulated and 293B-inhibited K+ conductance of rat colonic crypt base cells. Pflugers Arch. 1996 May;432(1):81–88. doi: 10.1007/s004240050108. [DOI] [PubMed] [Google Scholar]
  50. Weuta H. Clinical studies with oral clotrimazole. Postgrad Med J. 1974 Jul;50 (Suppl 1):45–48. [PubMed] [Google Scholar]
  51. Wong S. M., Tesfaye A., DeBell M. C., Chase H. S., Jr Carbachol increases basolateral K+ conductance in T84 cells. Simultaneous measurements of cell [Ca] and gK explore calcium's role. J Gen Physiol. 1990 Dec;96(6):1271–1285. doi: 10.1085/jgp.96.6.1271. [DOI] [PMC free article] [PubMed] [Google Scholar]

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