Abstract
The present study investigates the mechanisms controlling tight junction permeability of the tracheal epithelium, with an emphasis on the regulatory role of intra- and extracellular calcium as well as the cell cytoskeleton. The tracheas were isolated from rabbits and their junctional permeability barrier was investigated in vitro by means of transepithelial electrical resistance measurements and flux measurements of the radiolabeled paracellular tracer, 14C-mannitol. The effects of intra- and extracellular calcium were studied using the calcium ionophore A 23187 and EGTA, and that of the cytoskeleton was investigated using cytochalasin B. Intracellular calcium of the tracheal epithelium was monitored microfluorometrically using the specific calcium indicator, Fura-2 AM (acetoxymethyl ester). The results indicate that the tight junction permeability of the trachea was significantly increased upon treatment with all three of the test compounds, as evidenced by a substantial decrease in transepithelial electrical resistance and an increase in transepithelial flux of 14C-mannitol. The effects of EGTA and cytochalasin B on the tight junction permeability are fully reversible upon removal of the compounds from the bathing media. On the other hand, tissues treated with the calcium ionophore demonstrate a partial or no recovery in membrane permeability, depending on the intracellular calcium levels. Moderate and transient increases in intracellular calcium caused a partial reversibility of the membrane resistance, while high and sustained intracellular calcium levels induce a complete irreversibility of the membrane resistance. These results suggest that high extracellular calcium levels and low intracellular calcium levels are required for the normal maintenance of the junctional permeability in the tracheal epithelium. Studies using cytochalasin B indicate that there is also a close relationship between the tight junctions and the organization of actin microfilaments. Alterations of these structures as well as cellular calcium levels can result in a substantial change in transepithelial permeability. Therefore compounds that affect tight junction permeability may exert their action through the calcium and cytoskeleton mechanisms.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
REFERENCES
A. Adjei and J. Garren. Pulmonary delivery of peptide drugs: Effect of particle size on bioavailability of leuprolide acetate in healthy human male volunteers. Pharm. Res. 7:565–569 (1990).
J. S. Patton, H. J. Fuchs, and J. A. Moore. Pulmonary delivery of peptide and protein drugs. Proc. 17th Int. Symp. Control. Release Soc., 1990, p. 214.
F. M. Wigley, J. H. Londono, and S. H. Wood. Insulin across respiratory mucosae by aerosol delivery. Diabetes 20:552–556 (1971).
Y. Rojanasakul, L. Y. Wang, M. Bhat, D. D. Dlover, C. J. Malanga, and J. K. H. Ma. The transport barrier of epithelia: A comparative study on membrane permeability and charge selectivity in the rabbit. Pharm. Res. 9:1029–1034 (1992).
D. W. Powell. Barrier function of epithelia. Am. J. Physiol. 241:G275 (1981).
M. Vidgren, P. Paronen, P. Vidgren, P. Vainio, and J. Nuutinen. Radiotracer evaluation of the drug particles inhaled from a new powder inhaler. Int. J. Pharm. 64:1–6 (1990).
L. Labedzki, J. M. Scavone, H. R. Ochs, and D. J. Greenblatt. Reduced systemic absorption of intrabronchial lidocaine by high-frequency nebulization. J. Clin. Pharmacol. 30:795–797 (1990).
J. Richardson, T. Bouchard, and C. C. Ferguson. Uptake and transport of exogeneous proteins by respiratory epithelium. Lab. Invest. 35:307–314 (1976).
J. L. Madara, M. R. Neutra, and J. S. Trier. Junctional complexes in fetal rat intestine during morphogenesis. Dev. Biol. 86:170–178 (1981).
P. M. Elias and D. S. Friend. Vitamin A induced mucous metaplasia. An in vitro system for modulating tight and gap junction differentiation. J. Cell Biol. 68:173–188 (1976).
D. K. Bhalla, S. M. Lavan, and C. Crocker. Airway permeability in rats exposed to ozone or treated with cytoskeleton-destabilizing drugs. Exp. Lung Res. 14:501–525 (1988).
G. K. Ojakian. Tumor promoter-induced changes in permeability of epithelial cell tight junctions. Cell 23:95–103 (1981).
I. Meza, G. Ibarra, M. Sabanero, A. Martinez-Palomo, and M. Cereijido. Occluding junctions and cytoskeletal components in a cultured transporting epithelium. J. Cell Biol. 87:746–754 (1980).
J. L. Madara. Intestinal absorptive cell tight junctions are linked to cytoskeleton. Am. J. Physiol. 253:C171–C175 (1987).
Y. Rojanasakul and J. R. Robinson. Electrophysiological and ultrastructural characterization of the cornea during in vitro perfusion. Int. J. Pharm. 63:1–16 (1990).
G. Grynkiewicz, M. Peonie, and R. Y. Tsien. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J. Biol. Chem. 260:3440–3450 (1985).
M. Cerijido, J. Ehrenfield, I. Meza, and A. Martinez-Palomo. Structural and functional membrane polarity in cultured monolayers of MDCK cells. J. Membr. Biol. 52:147–159 (1980).
B. Gumbiner. Structure, biochemistry, and assembly of epithelial tight junctions. Am. J. Physiol. 253:C749–C758 (1987).
B. R. Stevenson and D. A. Goodenough. Zonulae occludentes in functional complex-enriched fractions from mouse liver: Preliminary morphological and biochemical characterization. J. Cell Biol. 98:1209–1221 (1984).
J. L. Madara, D. Barenberg, and S. Carlson. Effects of cytochalasin D on occluding junctions of intestinal absorptive cells: Further evidence that the cytoskeleton may influence paracellular permeability and junctional charge selectivity. J. Cell Biol. 97:125–136 (1983).
S. S. Brown and J. A. Spudich. Mechanism of action of cytochalasin: Evidence that it binds to actin filament ends. J. Cell Biol. 88:487–491 (1981).
J. L. Madara, J. Stafford, D. Barenberg, and S. Carlson. Functional coupling of tight junctions and microfilaments in T84 monolayers. Am. J. Physiol. 254:G416–G423 (1988).
C. E. Palant, M. E. Duffey, B. K. Mookerjee, S. Ho, and C. J. Bentzel. Ca2+ regulation of tight-junction permeability and structure in Necturus gallbladder. Am. J. Physiol. 245:C203–C212 (1983).
D. R. Pitelka and B. N. Taggart. Mechanical tension induces lateral movement of intramembrane components of the tight junction: Studies on mouse mammary cells in culture. J. Cell Biol. 96:606–612 (1983).
A. Martinez-Palomo, I. Meza, G. Beaty, and M. Cereijido. Experimental modulation of occluding junctions in a cultured transporting epithelium. J. Cell Biol. 87:736–745 (1980).
M. J. Rutten, N. Cogburn, C. S. Schasteen, and T. Solomon. Physiological and cytotoxic effects of Ca2+ ionophores on Caco-2 paracellular permeability: Relationship of 45Ca2+ efflux to 51Cr release. Pharmacology 42:156–168 (1991).
P. Artursson and C. Magnusson. Epithelial transport of drugs in cell culture. II. Effect of extracellular calcium concentration on the paracellular transport of drugs of different lipophilicities across monolayers of intestinal epithelial (Caco-2) cells. J. Pharm. Sci. 79:595–600 (1990).
M. S. Mooseker, T. R. Coleman, and K. A. Conzelman. Calcium and the regulation of cytoskeletal assembly, structure and contractility. In Calcium and the Cell, Wiley, Chichester (Ciba Foundation Symposium 122), 1986, pp. 232–249.
K. L. Rice, P. G. Duane, G. Mielke, A. A. Sinha, and D. E. Niewoehner. Calcium ionophores injure alveolar epithelial cells: Relation to phospholipase activity. Am. J. Physiol. 259:L439–L450 (1990).
R. P. Schmid, D. Wangensteen, J. Hoidal, B. Gosnell, and D. Niewoehner. Effects of elastase and cigarette smoke on alveolar epithelial permeability. J. Appl. Physiol. 59:96–100 (1985).
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Bhat, M., Toledo-Velasquez, D., Wang, L. et al. Regulation of Tight Junction Permeability by Calcium Mediators and Cell Cytoskeleton in Rabbit Tracheal Epithelium. Pharm Res 10, 991–997 (1993). https://doi.org/10.1023/A:1018906504944
Issue Date:
DOI: https://doi.org/10.1023/A:1018906504944