SMOKELESS TOBACCO-EXPOSED ORAL KERATINOCYTES INCREASE MACROMOLECULAR EFFLUX FROM THE IN-SITU ORAL-MUCOSA

The purpose of this study was to determine whether supernatants of cul tured human oral keratinocytes (HOK) exposed to an aqueous extract of smokeless tobacco (STE) increase macromolecular efflux from the oral m ucosa in vivo and, if so, whether bradykinin mediates in part this res ponse. Subconfluent monolayers of HOK were incubated with STE or media , and supernatants were collected 24, 48, and 72 h thereafter. Using i ntravital microscopy, we found that suffusion of supernatants of STE-b ut not media-exposed HOK elicited significant concentration-and time-d ependent increases in efflux of fluorescein isothiocyanate-labeled dex tran (mol mass 70 kDa) from the in situ hamster cheek pouch (P < 0.05) . These effects were significantly attenuated by HOE-140 and NPC-17647 but not by des-Arg(9),[Leu(8)]-bradykinin. Proteolytic activity was i ncreased in supernatants of STE-but not media-exposed HOK. However, a mixture of leupeptin, Bestatin, and DL-2-mercaptomethyl-3-guanidinoeth ylthiopropanoic acid had no significant effects on HOK supernatant-ind uced responses. Collectively, these data suggest that oral keratinocyt es modulate smokeless tobacco-induced increase in macromolecular efflu x from the in situ oral mucosa in part by elaborating proteases that m ay account for local bradykinin production.