Skip to main content
SpringerLink
Log in

Enhancement of Insulin Transport Across Primary Rat Alveolar Epithelial Cell Monolayers by Endogenous Cellular Factor(s)

  • Research Paper
  • Published:
Pharmaceutical Research Aims and scope Submit manuscript

Abstract

Purpose

To characterize factor(s) contained in apical medium of primary cultured rat alveolar epithelial type II cell-like monolayers (RAECM-II) that enhance insulin absorption across alveolar epithelial cells.

Materials and Methods

Primary rat alveolar epithelial cell monolayers cultured on Transwells in the presence and absence of 10 ng/ml keratinocyte growth factor for 6 days were dosed from the apical compartment with radiolabeled insulin in: newborn bovine serum-containing medium (SM), conditioned medium from apical compartment of rat alveolar epithelial type I cell-like monolayers (RAECM-I) (CMI), or conditioned medium from apical compartment of RAECM-II (CMII). At the end of 2 h incubation, basolateral medium was collected and amounts of transported radiolabeled insulin were determined using a gamma counter. In order to determine the molecular size range of the enhancing factor(s), CMII was centrifuged in 50 kDa molecular weight cut-off Centricon tubes, and both retentate and filtrate were used as separate dosing solutions. Heat denaturation and ammonium sulphate precipitation were used to determine if the involved factor(s) represent proteins or other smaller soluble factors. Transalveolar transport rates of a paracellular marker, 14C-mannitol, and fluid-phase marker, horseradish peroxidase, were determined in the presence and absence of the factors. Effects of temperature (4, 16 and 37°C) on radiolabeled insulin fluxes were also measured.

Results

Conditioned medium obtained from the apical compartment of RAECM-II, CMII, increased transport of insulin across the monolayers when compared to SM or CMI. The enhancing effect of CMII was retained in the precipitate following ammonium sulfate treatment and in the retentate after Centricon filtration. The enhancing effect of CMII was significantly decreased when heated at 80°C for 15 min. CMII did not affect the transport of 14C-mannitol or HRP, while its effect on insulin transport was decreased by 87% when temperature was lowered to 4°C from 37°C.

Conclusions

Conditioned medium from type II cell-like monolayer cultures appears to contain protein factor(s) which seem to be involved in facilitating active transcellular transport of insulin across primary cultured RAECM-II.

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

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  1. J. S. Patton, J. Bukar, and S. Nagarajan. Inhaled insulin. Adv. Drug Deliv. Rev. 35:235–247 (1999).

    Article  PubMed  CAS  Google Scholar 

  2. F. W. Wigley, J. H. Londono, S. H. Wood, J. C. Shipp, and R. H. Waldman. Insulin across respiratory mucosae by aerosol delivery. Diabetes 20:552–556 (1971).

    PubMed  CAS  Google Scholar 

  3. J. S. Patton, C. S. Fishburn, and J. G. Weers. The lungs as a portal of entry for systemic drug delivery. Proc. Am. Thorac. Soc. 1:338–344 (2004).

    Article  PubMed  CAS  Google Scholar 

  4. J. S. Patton. Mechanisms of macromolecule absorption by the lungs. Adv. Drug Deliv. Rev. 19:3–36 (1996).

    Article  CAS  Google Scholar 

  5. A. Yamamoto, S. Umemori, S. Muranishi and S. Muranishi. Absorption enhancement of intrapulmonary administered insulin by various absorption enhancers and protease inhibitors in rats. J. Pharm. Pharmacol. 46:14–18 (1994).

    PubMed  CAS  Google Scholar 

  6. F. Komada, S. Iwakawa, F. N. Yamamoto, H. Sakakibara, and K. Okumura. Intratracheal delivery of insulin: absorption from solution and aerosol by rat lung. Int. J. Pharm. 88:63–73 (1992).

    Article  Google Scholar 

  7. F. Johansson, E. Hjertberg, S. Eirefelt, A. Tronde, and U. Hultkvist Bengtsson. Mechanisms for absorption enhancement of inhaled insulin by sodium taurocholate. Eur. J. Pharm. Sci. 17:63–71 (2002).

    Article  PubMed  CAS  Google Scholar 

  8. L. Li, N. R. Mathias, C. L. Heran, P. Moench, D. A. Wall, and R. L. Smith. Carbopol-mediated paracellular transport enhancement in Calu-3 cell layers. J. Pharm. Sci. 95:326–335 (2006).

    Article  PubMed  CAS  Google Scholar 

  9. Y. Fukuda, T. Tsuji, T. Fujita, A. Yamamoto, and S. Muranishi. Susceptibility of insulin to proteolysis in rat lung homogenate and its protection from proteolysis by various protease inhibitors. Biol. Pharm. Bull. 18:891–894 (1995).

    PubMed  CAS  Google Scholar 

  10. Z. Shen, Q. Zhang, S. Wei, and T. Nagai. Proteolytic enzymes as a limitation for pulmonary absorption of insulin: in vitro and in vivo investigations. Int. J. Pharm. 192:115–121 (1999).

    Article  PubMed  CAS  Google Scholar 

  11. S. Kobayashi, S. Kondo, and K. Juni. Study on pulmonary delivery of salmon calcitonin in rats: effects of protease inhibitors and absorption enhancers. Pharm. Res. 11:239–1243 (1994).

    Google Scholar 

  12. A. J. Bitonti, J. A. Dumont, S. C. Low, R. T. Peters, K. E. Kropp, V. J. Palombella, J. M. Stattel, Y. Lu, C. A. Tan, J. J. Song, A. M. Garcia, N. E. Simister, G. M. Spiekermann, W. I. Lencer, and R. S. Blumberg. Pulmonary delivery of an erythropoietin Fc fusion protein in non-human primates through an immunoglobulin transport pathway. Proc. Natl. Acad. Sci. USA 101:9763–9768 (2004).

    Article  PubMed  CAS  Google Scholar 

  13. A. J. Bitonti and J. A. Dumont. Pulmonary administration of therapeutic proteins using an immunoglobulin transport pathway. Adv. Drug Deliv. Rev. 58:1106–1118 (2006).

    Article  PubMed  CAS  Google Scholar 

  14. A. Widera, K. J. Kim, E. D. Crandall, and W. C. Shen. Transcytosis of GCSF-transferrin across rat alveolar epithelial cell monolayers. Pharm. Res. 20:1231–1238 (2003).

    Article  PubMed  CAS  Google Scholar 

  15. D. Deshpande, D. Toledo-Velasquez, L. Y. Wang, C. J. Malanga, J. K. Ma, and Y. Rojanasakul. Receptor-mediated peptide delivery in pulmonary epithelial monolayers. Pharm. Res. 11:1121–1126 (1994).

    Article  PubMed  CAS  Google Scholar 

  16. M. E. Brandes and J. N. Finkelstein. Stimulated rabbit alveolar macrophages secrete a growth factor for type II pneumocytes. Am. J. Respir. Cell Mol. Biol. 1:101–109 (1989).

    PubMed  CAS  Google Scholar 

  17. Y. Matsukawa, H. Yamahara, F. Yamashita, V. H. Lee, E. D. Crandall, and K. J. Kim. Rates of protein transport across rat alveolar epithelial cell monolayers. J. Drug Target. 7:335–342 (2000).

    Article  PubMed  CAS  Google Scholar 

  18. M. Bur, H. Huwer, C. M. Lehr, N. Hagen, M. Guldbrandt, K. J. Kim, and C. Ehrhardt. Assessment of transport rates of proteins and peptides across primary human alveolar epithelial cell monolayers. Eur. J. Pharm. Sci. 28:196–203 (2006).

    Article  PubMed  CAS  Google Scholar 

  19. Z. Borok, R. L. Lubman, S. I. Danto, X. L. Zhang, S. M. Zabski, L. S. King, D. M. Lee, P. Agre, and E. D. Crandall. Keratinocyte growth factor modulates alveolar epithelial cell phenotype in vitro: expression of aquaporin 5. Am. J. Respir. Cell Mol. Biol. 18:554–561 (1998).

    PubMed  CAS  Google Scholar 

  20. R. Mitra, I. Pezron, Y. Li, and A. K. Mitra. Enhanced pulmonary delivery of insulin by lung lavage fluid and phospholipids. Int. J. Pharm. 217:25–31 (2001).

    Article  PubMed  CAS  Google Scholar 

  21. P. J. McConahey and F. J. Dixon. Radioiodination of proteins by the use of the chloramine-T method. Methods Enzymol. 70:210–213 (1980).

    Article  PubMed  CAS  Google Scholar 

  22. V. Herzog and H. D. Fahimi. A new sensitive colorimetric assay for peroxidase using 3,3′-diaminobenzidine as hydrogen donor. Anal. Biochem. 55:554–562 (1973).

    Article  PubMed  CAS  Google Scholar 

  23. A. Hussain, J. J. Arnold, M. A. Khan, and F. Ahsan. Absorption enhancers in pulmonary protein delivery. J. Control. Release 94:15–24 (2004).

    Article  PubMed  CAS  Google Scholar 

  24. T. Y. Ma, D. Nguyen, V. Bui, H. Nguyen, and N. Hoa. Ethanol modulation of intestinal epithelial tight junction barrier. Am. J. Physiol. 276:G965–G974 (1999).

    PubMed  CAS  Google Scholar 

  25. V. A. K. Dodane, M. Amin Khan, and J. R. Merwin. Effect of chitosan on epithelial permeability and structure. Int. J. Pharm. 10:21–32 (1999).

    Article  Google Scholar 

  26. M. Machida, M. Hayashi, and S. Awazu. The effects of absorption enhancers on the pulmonary absorption of recombinant human granulocyte colony-stimulating factor (rhG-CSF) in rats. Biol. Pharm. Bull. 23:84–86 (2000).

    PubMed  CAS  Google Scholar 

  27. T. Morita, A. Yamamoto, Y. Takakura, M. Hashida, and H. Sezaki. Improvement of the pulmonary absorption of (Asu1,7)-eel calcitonin by various protease inhibitors in rats. Pharm. Res. 11:909–913 (1994).

    Article  PubMed  CAS  Google Scholar 

  28. B. Forbes, C. G. Wilson, and M. Gumbleton. Temporal dependence of ectopeptidase expression in alveolar epithelial cell culture: implications for study of peptide absorption. Int. J. Pharm. 180:225–234 (1999).

    Article  PubMed  CAS  Google Scholar 

  29. M. C. Hsu and J. P. Bai. Investigation into the presence of insulin-degrading enzyme in cultured type II alveolar cells and the effects of enzyme inhibitors on pulmonary bioavailability of insulin in rats. J. Pharm. Pharmacol. 50:507–514 (1998).

    PubMed  CAS  Google Scholar 

  30. S. Y. Hirai, T. Yashiki, and H. Mima. Mechanisms for the enhancement of the nasal absorption of insulin by surfactants. Int. J. Pharm. 9:173–184 (1981).

    Article  CAS  Google Scholar 

  31. G. S. Gordon, A. C. Moses, R. D. Silver, J. S. Flier, and M. C. Carey. Nasal absorption of insulin: enhancement by hydrophobic bile salts. Proc. Natl. Acad. Sci. USA 82:7419–7423 (1985).

    Article  PubMed  CAS  Google Scholar 

  32. R. H. Hastings, H. G. Folkesson, and M. A. Matthay. Mechanisms of alveolar protein clearance in the intact lung. Am. J. Physiol. 286:L679–L689 (2004).

    CAS  Google Scholar 

  33. D. Shah and W. C. Shen. The paradox of transferrin receptor-mediated drug delivery-intracellular targeting or transcellular transport? J. Drug Target. 3:243–245 (1995).

    PubMed  CAS  Google Scholar 

  34. L. G. Dobbs, M. C. Williams, and A. E. Brandt. Changes in biochemical characteristics and pattern of lectin binding of alveolar type II cells with time in culture. Biochim. Biophys. Acta 846:155–166 (1985).

    Article  PubMed  CAS  Google Scholar 

  35. M. Sakagami. In vivo, in vitro and ex vivo models to assess pulmonary absorption and disposition of inhaled therapeutics for systemic delivery. Adv. Drug Deliv. Rev. 58:1030–1060 (2006).

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

We thank Ray Alvarez for preparing freshly isolated alveolar epithelial type II cells for this study. This work was supported by research grants HL64365, HL62569, HL38578, HL38621, and HL38658 from the National Institutes of Health and the Hastings Foundation. E. D. C. is Kenneth T. Norris Chair and Hastings Professor of Medicine.

Author information

Authors and Affiliations

  1. Department of Pharmacology and Pharmaceutical Sciences, University of Southern California, Los Angeles, California, 90089-9121, USA

    Rana Bahhady & Wei-Chiang Shen

  2. Department of Medicine, University of Southern California, Los Angeles, California, 90089-9121, USA

    Kwang-Jin Kim, Zea Borok & Edward D. Crandall

  3. Will Rogers Institute Pulmonary Research Center, University of Southern California, Los Angeles, California, 90089-9121, USA

    Kwang-Jin Kim, Zea Borok & Edward D. Crandall

Authors
  1. Rana Bahhady
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kwang-Jin Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Zea Borok
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Edward D. Crandall
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Wei-Chiang Shen
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Wei-Chiang Shen.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Bahhady, R., Kim, KJ., Borok, Z. et al. Enhancement of Insulin Transport Across Primary Rat Alveolar Epithelial Cell Monolayers by Endogenous Cellular Factor(s). Pharm Res 24, 1713–1719 (2007). https://doi.org/10.1007/s11095-007-9301-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11095-007-9301-9

Key words

Search

Navigation