Poly-L-arginine predominantly increases the paracellular permeability of hydrophilic macromolecules across rabbit nasal epithelium in vitro.
OBJECTIVE
The purpose of this work was to characterize the main transport pathway of hydrophilic macromolecules induced by poly-L-arginine (poly-L-Arg; molecular weight 42.4 kDa) across the excised rabbit nasal epithelium.


METHODS
Excised rabbit nasal epithelium was mounted in an Ussing-type chamber for measurement of fluorescein isothiocyanate-labeled dextran (FD-4; molecular weight 4.4 kDa) transport and transepithelial electrical resistance (TEER). The main transport pathway of FD-4 enhanced by poly-L-Arg was evaluated using confocal laser scanning microscopy. Immunolocalization of junction proteins (ZO-1, occludin, and E-cadherin) after treatment with poly-L-Arg was also observed.


RESULTS
After apical application of a poly-L-Arg (0.05, 0.5, and 5 mg/mL), the permeability coefficient of FD-4 increased by 1.6-, 2.9-, and 5.2-fold, respectively, compared with the control of 5.2 +/- 1.3 x 10(-7) cm/s. Consistent with the increase in transport, there was a concurrent reduction in TEER. At a concentration of 0.05 mg/mL poly-L-Arg. both FD-4 transport and TEER returned to the control level. A good correlation was obtained between the FD-4 permeability coefficient and 1/TEER. Basolateral application of poly-L-Arg at 5 mg/mL, however, did not increase FD-4 transport. Marked FD-4 fluorescence was located in the paracellular spaces after treatment with apical poly-L-Arg compared with that in the absence of poly-L-Arg. Immunofluorescence of ZO-1, occludin, and E-cadherin in cell-to-cell junctions was reduced and distributed into the cytoplasm by apical application of poly-L-Arg, suggesting that poly-L-Arg regulates the junction proteins to enhance paracellular permeability across the nasal epithelium. After pretreatment with either 2,4-dinitrophenol or ouabain, the enhancing effect of apical poly-L-Arg was abolished, indicating the contribution of metabolic energy (cell viability) to the poly-L-Arg-mediated enhancing effect.


CONCLUSIONS
In the nasal epithelium, apical poly-L-Arg appears to increase predominantly the paracellular transport of hydrophilic macromolecules via disorganization of tight- and adherens-junction proteins. The regulatory mechanism of the poly-L-Arg effect is likely to be dependent on energy-requiring cellular processes.
Ohtake
Kazuo
K
Faculty of Pharmaceutical Sciences, Josai University, 1-1 Keyakidai, Sakado, Saitama 350-0295, Japan.
Maeno
Takaya
T
Ueda
Hideo
H
Natsume
Hideshi
H
Morimoto
Yasunori
Y
D000818:Animals; D001692:Biological Transport / Q000187:drug effects / Q000502:physiology; D004357:Drug Synergism; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D008297:Male; D009297:Nasal Mucosa / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D010455:Peptides / Q000493:pharmacokinetics*; D010539:Permeability / Q000187:drug effects; D011817:Rabbits
D046911:Macromolecular Substances; D010455:Peptides; C015462:polyarginine