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Divergent Inhibitor Susceptibility among Airway Lumen-Accessible Tryptic Proteases.

Nimishakavi S, Raymond WW, Gruenert DC, Caughey GH - PLoS ONE (2015)

Bottom Line: We hypothesized that these inhibitors inactivate a variety of airway protease targets, potentially with bystander effects.Aprotinin exhibited nearly stoichiometric inhibition of prostasin and matriptase, but was much weaker towards HAT and was completely ineffective versus tryptase.Nafamostat, camostat and aprotinin markedly reduced tryptic activity on the apical surface of cystic fibrosis airway epithelial monolayers, suggesting prostasin as the major source of such activity and supporting strategies targeting prostasin for inactivation.

View Article: PubMed Central - PubMed

Affiliation: Cardiovascular Research Institute, University of California San Francisco, San Francisco, California, United States of America; Veterans Affairs Medical Center, San Francisco, California, United States of America.

ABSTRACT
Tryptic serine proteases of bronchial epithelium regulate ion flux, barrier integrity, and allergic inflammation. Inhibition of some of these proteases is a strategy to improve mucociliary function in cystic fibrosis and asthmatic inflammation. Several inhibitors have been tested in pre-clinical animal models and humans. We hypothesized that these inhibitors inactivate a variety of airway protease targets, potentially with bystander effects. To establish relative potencies and modes of action, we compared inactivation of human prostasin, matriptase, airway trypsin-like protease (HAT), and β-tryptase by nafamostat, camostat, bis(5-amidino-2-benzimidazolyl)methane (BABIM), aprotinin, and benzamidine. Nafamostat achieved complete, nearly stoichiometric and very slowly reversible inhibition of matriptase and tryptase, but inhibited prostasin less potently and was weakest versus HAT. The IC50 of nafamostat's leaving group, 6-amidino-2-naphthol, was >104-fold higher than that of nafamostat itself, consistent with suicide rather than product inhibition as mechanisms of prolonged inactivation. Stoichiometric release of 6-amidino-2-naphthol allowed highly sensitive fluorometric estimation of active-site concentration in preparations of matriptase and tryptase. Camostat inactivated all enzymes but was less potent overall and weakest towards matriptase, which, however was strongly inhibited by BABIM. Aprotinin exhibited nearly stoichiometric inhibition of prostasin and matriptase, but was much weaker towards HAT and was completely ineffective versus tryptase. Benzamidine was universally weak. Thus, each inhibitor profile was distinct. Nafamostat, camostat and aprotinin markedly reduced tryptic activity on the apical surface of cystic fibrosis airway epithelial monolayers, suggesting prostasin as the major source of such activity and supporting strategies targeting prostasin for inactivation.

No MeSH data available.


Related in: MedlinePlus

Inhibition of surface tryptic activity on bronchial epithelial cells.Monolayers of CFBE41o- cells cultured on Transwell inserts at an air-liquid interface to promote differentiation, including apical-basolateral polarization, were assayed for surface tryptic activity by adding substrate QAR to apical bathing medium, with or without addition of camostat, nafamostat or aprotinin, followed by spectrophotometric monitoring of cleaved substrate at 410 nm. *P <0.05 and **P <0.01 versus change in absorbance in QAR medium without inhibitor.
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pone.0141169.g007: Inhibition of surface tryptic activity on bronchial epithelial cells.Monolayers of CFBE41o- cells cultured on Transwell inserts at an air-liquid interface to promote differentiation, including apical-basolateral polarization, were assayed for surface tryptic activity by adding substrate QAR to apical bathing medium, with or without addition of camostat, nafamostat or aprotinin, followed by spectrophotometric monitoring of cleaved substrate at 410 nm. *P <0.05 and **P <0.01 versus change in absorbance in QAR medium without inhibitor.

Mentions: Fig 7 reveals that apical, cell surface, QAR-hydrolyzing protease activity is almost completely inhibited by aprotinin (100 μM), consistent with our prior study [3], while establishing that nafamostat and camostat (10 μM) are nearly as effective as aprotinin. In conjunction with our prior studies showing little if any active matriptase on the apical surface of CFBE41o- cells cultured in the same manner [3], the present data are consistent with prostasin (and not HAT or β-tryptase) being the principal source of apical tryptic activity.


Divergent Inhibitor Susceptibility among Airway Lumen-Accessible Tryptic Proteases.

Nimishakavi S, Raymond WW, Gruenert DC, Caughey GH - PLoS ONE (2015)

Inhibition of surface tryptic activity on bronchial epithelial cells.Monolayers of CFBE41o- cells cultured on Transwell inserts at an air-liquid interface to promote differentiation, including apical-basolateral polarization, were assayed for surface tryptic activity by adding substrate QAR to apical bathing medium, with or without addition of camostat, nafamostat or aprotinin, followed by spectrophotometric monitoring of cleaved substrate at 410 nm. *P <0.05 and **P <0.01 versus change in absorbance in QAR medium without inhibitor.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4612780&req=5

pone.0141169.g007: Inhibition of surface tryptic activity on bronchial epithelial cells.Monolayers of CFBE41o- cells cultured on Transwell inserts at an air-liquid interface to promote differentiation, including apical-basolateral polarization, were assayed for surface tryptic activity by adding substrate QAR to apical bathing medium, with or without addition of camostat, nafamostat or aprotinin, followed by spectrophotometric monitoring of cleaved substrate at 410 nm. *P <0.05 and **P <0.01 versus change in absorbance in QAR medium without inhibitor.
Mentions: Fig 7 reveals that apical, cell surface, QAR-hydrolyzing protease activity is almost completely inhibited by aprotinin (100 μM), consistent with our prior study [3], while establishing that nafamostat and camostat (10 μM) are nearly as effective as aprotinin. In conjunction with our prior studies showing little if any active matriptase on the apical surface of CFBE41o- cells cultured in the same manner [3], the present data are consistent with prostasin (and not HAT or β-tryptase) being the principal source of apical tryptic activity.

Bottom Line: We hypothesized that these inhibitors inactivate a variety of airway protease targets, potentially with bystander effects.Aprotinin exhibited nearly stoichiometric inhibition of prostasin and matriptase, but was much weaker towards HAT and was completely ineffective versus tryptase.Nafamostat, camostat and aprotinin markedly reduced tryptic activity on the apical surface of cystic fibrosis airway epithelial monolayers, suggesting prostasin as the major source of such activity and supporting strategies targeting prostasin for inactivation.

View Article: PubMed Central - PubMed

Affiliation: Cardiovascular Research Institute, University of California San Francisco, San Francisco, California, United States of America; Veterans Affairs Medical Center, San Francisco, California, United States of America.

ABSTRACT
Tryptic serine proteases of bronchial epithelium regulate ion flux, barrier integrity, and allergic inflammation. Inhibition of some of these proteases is a strategy to improve mucociliary function in cystic fibrosis and asthmatic inflammation. Several inhibitors have been tested in pre-clinical animal models and humans. We hypothesized that these inhibitors inactivate a variety of airway protease targets, potentially with bystander effects. To establish relative potencies and modes of action, we compared inactivation of human prostasin, matriptase, airway trypsin-like protease (HAT), and β-tryptase by nafamostat, camostat, bis(5-amidino-2-benzimidazolyl)methane (BABIM), aprotinin, and benzamidine. Nafamostat achieved complete, nearly stoichiometric and very slowly reversible inhibition of matriptase and tryptase, but inhibited prostasin less potently and was weakest versus HAT. The IC50 of nafamostat's leaving group, 6-amidino-2-naphthol, was >104-fold higher than that of nafamostat itself, consistent with suicide rather than product inhibition as mechanisms of prolonged inactivation. Stoichiometric release of 6-amidino-2-naphthol allowed highly sensitive fluorometric estimation of active-site concentration in preparations of matriptase and tryptase. Camostat inactivated all enzymes but was less potent overall and weakest towards matriptase, which, however was strongly inhibited by BABIM. Aprotinin exhibited nearly stoichiometric inhibition of prostasin and matriptase, but was much weaker towards HAT and was completely ineffective versus tryptase. Benzamidine was universally weak. Thus, each inhibitor profile was distinct. Nafamostat, camostat and aprotinin markedly reduced tryptic activity on the apical surface of cystic fibrosis airway epithelial monolayers, suggesting prostasin as the major source of such activity and supporting strategies targeting prostasin for inactivation.

No MeSH data available.


Related in: MedlinePlus