Pharmaceutical Research

, Volume 23, Issue 3, pp 540–548 | Cite as

Voltage Dependence of Transepithelial Guanidine Permeation Across Caco-2 Epithelia Allows Determination of the Paracellular Flux Component

  • Georgina Carr
  • Iain S. Haslam
  • Nicholas L. Simmons
Research Paper
First Online:
Received:
Accepted:

Purpose

The aim of this study was to investigate transepithelial ionic permeation via the paracellular pathway of human Caco-2 epithelial monolayers and its contribution to absorption of the base guanidine.

Methods

Confluent monolayers of Caco-2 epithelial cells were mounted in Ussing chambers and the transepithelial conductance and electrical potential difference (p.d.) determined after NaCl dilution or medium Na substitution (bi-ionic conditions). Guanidine absorption (Ja–b) was measured ± transepithelial potential gradients using bi-ionic p.d.'s.

Results

Basal NaCl replacement with mannitol gives a transepithelial dilution p.d. of 28.0 ± 3.1 mV basal solution electropositive (PCl/PNa = 0.34). Bi-ionic p.d.'s (basal replacements) indicate a cation selectivity of NH4+ > K+∼Cs+ > Na+ > Li+ > tetraethylammonium+ > N-methyl-d-glucamine+∼choline+. Transepithelial conductances show good correspondence with bi-ionic potential data. Guanidine Ja–b was markedly sensitive to imposed transepithelial potential difference. The ratio of guanidine to mannitol permeability (measured simultaneously) increased from 3.6 in the absence of an imposed p.d. to 13.8 (basolateral negative p.d.).

Conclusions

Hydrated monovalent ions preferentially permeate the paracellular pathway (Eisenman sequence 2 or 3). Guanidine may access the paracellular pathway because absorptive flux is sensitive to the transepithelial potential difference. An alternative method to assess paracellular-mediated flux of charged organic molecules is suggested.

Key Words

Caco-2 cation selectivity human intestine organic cation absorption paracellular pathway 

Abbreviations

EVOM

epithelial volt-ohm meter

Ja-b

transepithelial flux from apical to basal bathing solution

Px

ionic permeability (for ion x)

Ψ, p.d.

transepithelial p.d. (mV)

Rt

transepithelial electrical resistance

This is a preview of subscription content, log in to check access.

Notes

Acknowledgments

Dr. C. Andersen supplied the Caco-2 epithelial layers (BBSRC grant 13/D17277). G. Carr performed the microscopy (Kidney Research UK). I. Haslam holds a BBSRC studentship.

References

  1. 1.
    Claude, P. 1978Morphological factors influencing transepithelial permeability: a model for the resistance of the zona occludensJ. Membr. Biol.10219232Google Scholar
  2. 2.
    Fromter, E. 1972The route of passive ion movement through the epithelium of Necturus gallbladderJ. Membr. Biol.8259301PubMedGoogle Scholar
  3. 3.
    Kottra, G., Fromter, E. 1983Functional properties of the paracellular pathway in some leaky epitheliaJ. Exp. Biol.106217229PubMedGoogle Scholar
  4. 4.
    Frizzell, R. A., Schultz, S. G. 1972Ionic conductances of extracellular shunt pathway in rabbit ileum. Influence of shunt on transmural sodium transport and electrical potential differencesJ. Gen. Physiol.59318346CrossRefPubMedGoogle Scholar
  5. 5.
    Schultz, S. G., Zalusky, R. 1964Ion transport in isolated rabbit ileum; short circuit current and Na-fluxesJ. Gen. Physiol.47567584PubMedGoogle Scholar
  6. 6.
    Simmons, N. L., Naftalin, R. J. 1976Bidirectional sodium ion movements via the paracellular and transcellular routes across short-circuited rabbit ileumBiochim. Biophys. Acta448426450PubMedGoogle Scholar
  7. 7.
    Artursson, P., Palim, K., Luthman, K. 2001Caco-2 monolayers in experimental and theoretical predictions of drug transportAdv. Drug Deliv. Rev.462743CrossRefPubMedGoogle Scholar
  8. 8.
    Cova, E., Laforenza, U., Gastaldi, G., Sambuy, Y., Tritto, S., Faelli, A., Ventura, U. 2002Guanidine transport across the apical and basolateral membranes of human intestinal Caco-2 cells is mediated by 2 different mechanismsJ. Nutr.13219952003PubMedGoogle Scholar
  9. 9.
    Thwaites, D. T., McEwan, G. T. A., Brown, C. D. A., Hirst, B. H., Simmons, N. L. 1993Na-independent, H-coupled transepithelial β-alanine absorption by human intestinal Caco-2 cell monolayersJ. Biol. Chem.2681843818441PubMedGoogle Scholar
  10. 10.
    Thwaites, D. T., Brown, C. D. A., Hirst, B. H., Simmons, N. L. 1993Transepithelial Gly-Sar transport in intestinal Caco-2 cells mediated by expression of H+-coupled carriers at both apical and basal membranesJ. Biol. Chem.26876407642PubMedGoogle Scholar
  11. 11.
    Thwaites, D. T., Cavet, M. G., Hirst, B. H., Simmons, N. L. 1995Angiotensin-converting enzyme (ACE) inhibitor transport in human intestinal epithelial (Caco-2) cellsBr. J. Pharmacol.114981986PubMedGoogle Scholar
  12. 12.
    Hille, B. 1972The permeability of the sodium channel to metal cations in myelinated nerveJ. Gen. Physiol.59637658CrossRefPubMedGoogle Scholar
  13. 13.
    Hunter, J., Hirst, B. H., Simmons, N. L. 1993Drug absorption limited by P-glycoprotein mediated secretory drug transport in human intestinal epithelial Caco-2 cell-layersPharm. Res.10743749CrossRefPubMedGoogle Scholar
  14. 14.
    Collett, A., Walker, D., Sims, E., He, Y. L., Speers, P., Ayrton, J., Rowland, M., Warhurst, G. 1997Influence of morphometric factors on quantification of paracellular permeability of intestinal epithelia in vitroPharm. Res.14767777CrossRefPubMedGoogle Scholar
  15. 15.
    Karlsson, J., Ungell, A.-L., Grasjo, J., Artursson, P. 1999Paracellular drug transport across intestinal epithelia: influence of charge and induced water fluxEur. J. Pharm. Sci.94756PubMedGoogle Scholar
  16. 16.
    Simon, D. B., Lu, Y., Choate, K. A., Velazquez, H., Al-Sabban, E., Praga, M., Casari, G., Bettinelli, A., Colussi, G., Rodriguez-Soriano, J., McCredie, D., Milford, D., Sanjad, S., Lifton, R. P. 1999Paracellin-1, a renal tight junction protein required for paracellular Mg2+ resorptionScience285103106CrossRefPubMedGoogle Scholar
  17. 17.
    Marin, M. L., Greenstein, A. J., Geler, S. A., Gordon, R. E., Aufses, A. H. 1983A freeze fracture study of Crohn's disease of the terminal ileum: changes in epithelial tight junction organisationAm. J. Gastroenterol.78537547PubMedGoogle Scholar
  18. 18.
    Soderholm, J. D., Olaison, G., Peterson, K. H., Franzen, L. E., Lindmark, T., Wiren, M., Tageson, C., Sjodahl, R. 2002Augmented increase in tight junction permeability by luminal stimuli in the non-inflamed ileum of Crohn's diseaseGut50307313CrossRefPubMedGoogle Scholar
  19. 19.
    Hyun, C. S., Chen, C. W., Shinowara, N. L., Paaia, T., Fallick, F. S., Martello, L. A., Muennnudde, M., Donovan, V. M., Teichberg, S. 1995Morphological factors influencing transepithelial conductance in a rabbit model of ileitisGastroenterology1091323CrossRefPubMedGoogle Scholar
  20. 20.
    Tavelin, S., Taipalensuu, J., Soderberg, L., Morrison, R., Chong, S., Artursson, P. 2003Prediction of the oral absorption of low-permeability drugs using an intestine-like 2/4/A1 cell monolayersPharm. Res.20397405PubMedGoogle Scholar
  21. 21.
    Adson, A., Burton, P. S., Raub, T. J., Barsuhn, C. L., Audus, K. L., Ho, B. F. 1995Passive diffusion of weak organic electrolytes across Caco-2 cell monolayers: uncoupling the contributions of hydrodynamic, transcellular and paracellular barriersJ. Pharm. Sci.84119711204PubMedGoogle Scholar
  22. 22.
    Watson, C. J., Rowl, M., Warhurst, G. 2001Functional modelling of tight junctions in intestinal cell monolayers using polyethylene glycol oligomersAm. J. Physiol.281C388C397Google Scholar
  23. 23.
    Nagahara, N., Tavelin, S., Artursson, P. 2004Contribution of the paracellular route to the pH-dependent epithelial permeability to cationic drugsJ. Pharm. Sci.9329722984CrossRefPubMedGoogle Scholar
  24. 24.
    Davis, G. R., Santa Ana, C. A., Morawski, S. G., Fordtran, J. S. 1982Permeability characteristics of human jejunum, ileum, proximal colon: results of potential difference measurements and unidirectional fluxesGastroenterology83844850PubMedGoogle Scholar
  25. 25.
    Gustke, R. F., McCormick, P., Ruppin, H., Soergel, K. H., Whalen, G. E., Wood, C. M. 1981Human intestinal potential difference: recording method and biophysical implicationsJ. Physiol.321571582PubMedGoogle Scholar
  26. 26.
    Lo, C.-M., Keese, C. R., Giaever, I. 1999Cell–substrate contact: another factor may influence transepithelial electrical resistance of cell layers cultured on permeable supportsExp. Cell Res.250576580CrossRefPubMedGoogle Scholar
  27. 27.
    Barry, P. H., Calc, J. P. 1994A software package for calculating liquid junction potential corrections in patch-clamp, intracellular, epithelial and bilayer measurements and for correcting junction potential measurementsJ. Neurosci. Methods51107116CrossRefPubMedGoogle Scholar
  28. 28.
    Grasset, E., Pinto, M., Dussaulx, E., Zweibaum, A., Desjeux, J.-F. 1984Epithelial properties of human colonic carcinoma cell line Caco-2: electrical parametersAm. J. Physiol.247C260C267PubMedGoogle Scholar
  29. 29.
    Eisenman, G., Horn, R. 1983Ion selectivity revisited: the role of kinetic and equilibrium processes in ion permeation through channelsJ. Membr. Biol.76197225PubMedGoogle Scholar
  30. 30.
    Cereijido, M., Robbins, E. S., Dolan, W. J., Rotunno, C. A., Sabatini, D. D. 1977Polarised monolayers formed by epithelial cells on a permeable and translucent supportJ. Cell Biol.77853879Google Scholar
  31. 31.
    Marcial, M. A., Carlson, S. L., Madara, J. L. 1984Partitioning of paracellular conductance along the crypt–villus axis: a hypothesis based on structural analysis with detailed consideration of tight-junction structure function relationshipsJ. Membr. Biol.805970PubMedGoogle Scholar
  32. 32.
    Stevenson, B. R., Anderson, J. M., Goodenough, D. A., Mooseker, M. S. 1988Tight junction structure and Zo-1 content are identical in two strains of Madin–Darby canine kidney cells which differ in transepithelial resistanceJ. Cell Biol.10724012408CrossRefPubMedGoogle Scholar
  33. 33.
    Tien, X.-Y., Brasitus, T. A., Kaetzel, M. A., Dedman, J. R., Nelson, D. J. 1994Activation of the cystic fibrosis transmembrane conductance regulator by cGMP in the human colonic cancer cell-line, Caco-2J. Biol. Chem.2695154PubMedGoogle Scholar
  34. 34.
    Tang, V. W., Goodenough, D. A. 2003Paracellular ion channel at the tight junctionBiophys. J.8416601673PubMedGoogle Scholar
  35. 35.
    Amasheh, S., Meiri, N., Gitter, A. H., Schoneberg, T., Mankertz, J., Schulz, J. D., Fromm, M. 2002Claudin-2 expression induces cation-selective channels in tight-junction of epithelial cellsJ. Cell Sci.1549694976Google Scholar
  36. 36.
    Van-Itlaie, C. M., Fanning, A. S., Anderson, J. M. 2003Reversal of charge selectivity in cation or anion-selective epithelial lines by expression of different claudinsAm. J. Physiol.285F1078F1084Google Scholar
  37. 37.
    Rahner, C., Mitic, L. L., Anderson, J. M. 2001Heterogeneity in expression and sub-cellular localization of claudins 2, 4 and 5 in the rat liver, pancreas and gutGastroenterology120411422CrossRefPubMedGoogle Scholar
  38. 38.
    McLaughlin, J., Padfield, P. J., Burt, J. P., O'Neil, C. A. 2004Ochratoxin A increases permeability through tight junctions by removal of specific claudin isoformsAm. J. Physiol.287C1412C1417CrossRefGoogle Scholar
  39. 39.
    Ginsburg, H., Stein, W. D. 2004The new permeability pathways induced by the malaria parasite in the membrane of the infected erythrocyte: comparison of results using different experimental techniquesJ. Membr. Biol.197113122CrossRefPubMedGoogle Scholar
  40. 40.
    Pade, V., Stavchansky, S. 1997Estimation of the relative contribution of the transcellular and paracellular pathway to the transport of passively absorbed drugs in the Caco-2 cell culture modelPharm. Res.1412101215CrossRefPubMedGoogle Scholar
  41. 41.
    Lee, K., Thakker, D. R. 1999Saturable transport of H2-antagonists ranitidine and famotidine across Caco-2 cell monolayersJ. Pharm. Sci.88680687PubMedGoogle Scholar

Copyright information

© Springer Science + Business Media, Inc. 2006

Authors and Affiliations

  • Georgina Carr
    • 1
  • Iain S. Haslam
    • 1
  • Nicholas L. Simmons
    • 1
  1. 1.Institute for Cell and Molecular Biosciences, Medical SchoolUniversity of Newcastle upon TyneNewcastle upon TyneUK

Personalised recommendations