Purified ACE attenuates smokeless tobacco-induced increase in macromolecular efflux from the oral mucosa

Abstract

Purified ACE attenuates smokeless tobacco-induced increase in macromolecular efflux from the oral mucosa. J. Appl. Physiol. 83(1): 74-81, 1997.-The purpose of this study was to determine whether purified angiotensin I-converting enzyme (ACE) attenuates smokeless tobacco extract (STE)-induced increase in macromolecular efflux from the in situ oral mucosa. By using intravital microscopy, we found that suffusion of an aqueous extract of smokeless tobacco elicited significant concentration-dependent leaky site formation and increase in clearance of fluorescein isothiocyanate-labeled dextran (mol mass, 70 kDa) from the hamster cheek pouch (P , 0.05). Suffusion of purified rabbit lung ACE significantly attenuated these responses in a concentration-dependent fashion (P , 0.05). These effects were specific because purified ACE also significantly attenuated the increase in macromolecular efflux elicited by bradykinin, which is produced in the cheek pouch during suffusion of STE, but did not attenuate the increase elicted by adenosine. Moreover, suffusion of heat-inactivated purified ACE and purified superoxide dismutase had no significant effects on STE-and bradykinin-induced responses. Collectively, these data suggest that exogenous ACE attenuates STE-induced increase in macromolecular efflux from the in situ oral mucosa, in part, by promoting local bradykinin catabolism.

Smokeless tobacco potentiates VIP-induced DNA synthesis and inactivates NEP 24.11 in oral keratinocytes

“…First, we found that exposure of HCPC-1 to aqueous STE, at noncytotoxic concentrations that have been previously shown to evoke inflammation in the in situ hamster cheek pouch (11,12), together with VIP, a ubiquitous autocrine neuropeptide in the oral epithelium (17), is associated with significant, concentration-dependent, synergistic increase in DNA synthesis as assessed by BrdU incorporation. These effects were mediated by VIP receptors because VIP 10-28 , a VIP receptor antagonist in the in situ hamster cheek pouch (43), but not VIP [1][2][3][4][5][6][7][8][9][10][11][12] , an inactive peptide fragment (43), significantly attenuated VIP-induced increase in DNA synthesis in the absence or presence of STE. Second, STE inactivated NEP 24.11, an ectoenzyme widely distributed in oral epithelium that cleaves and inactivates VIP very effectively (12,35,43,52), in HCPC-1 cells after 48-h incubation, a time when VIP-induced DNA synthesis was maximal.…”

“…Section 1734 solely to indicate this fact. previously described (11,12,27,37). Briefly, 10 g of smokeless tobacco (1S3 moist snuff; Tobacco and Health Research Institute, University of Kentucky, Lexington, KY) were mixed with 100 ml DMEM and incubated at 37°C for 2 h. The mixture was then centrifuged at 450 g for 10 min.…”

“…Briefly, 10 g of smokeless tobacco (1S3 moist snuff; Tobacco and Health Research Institute, University of Kentucky, Lexington, KY) were mixed with 100 ml DMEM and incubated at 37°C for 2 h. The mixture was then centrifuged at 450 g for 10 min. The supernatant was collected and centrifuged at 13,000 g for 1 h. After adjusting the pH to 7.4 using 0.1 N HCl, the resulting supernatant, designated arbitrarily as 1:10 aqueous dilution of raw smokeless tobacco (11,12,27,28,37), was filtered through a Millipore filter (pore size, 0.45 µm), divided into 2-ml samples, snap-frozen in liquid nitrogen, and stored at Ϫ70°C until used.…”

“…In some experiments, HCPC-1 cells were incubated with VIP 1-12 (10 Ϫ5 M), an inactive peptide fragment, alone and in the presence of VIP (10 Ϫ6 M) and VIP (10 Ϫ6 M) together with STE (1:100 aqueous dilution) for 24-72 h. Each experiment was conducted in triplicate. The concentrations of STE, VIP, VIP 10-28 , and VIP 1-12 used in these experiments are based on preliminary and previous studies in my laboratory and reports in the literature (11,12,27,28,37,39,43).…”

Subtilisin Increases Macromolecular Efflux from the Oral Mucosa

“…In preliminary studies, I determined that suffusion of aprotinin (5 g/ml) and leupeptin (10 g/ml) alone for 30 min was associated with no visible leaky site formation or increase in the clearance of FITC-dextran (data not shown). The concentrations of aprotinin and leupeptin used in these studies were based on previous studies in my laboratory and on reports in the literature (2,9,13,17,18,22,31,38).…”