Semester

Summer

Date of Graduation

2001

Document Type

Dissertation

Degree Type

PhD

College

School of Pharmacy

Department

Pharmaceutical Sciences

Committee Chair

Joseph K. H. Ma.

Abstract

Purpose. To demonstrate that alveolar macrophages (AM) from rats exhibit pepT1-like transporter for the uptake of small arginine-containing peptides (ACPs) and utilized these peptides as direct substrates for nitric oxide (NO) production.;Method. A HPLC assay was developed for quantitative measurement of Arg and ACPs in rat plasma and bronchoalveolar lavage (BAL) fluid. The uptake of small peptides by rat AM was evaluated using fluorescein isothiocynate (FITC)-labeled (*) peptides (Arg-Lys*, beta-Ala-Lys*, and Gly-Sar-Lys*), HPLC analysis of potential peptide degradation, and known inhibitors on arginine (Arg) and PepT1 transport. NO production by AM through Arg and ACPs was studied with and without inhibition by transport inhibitors. The presence of PepT1-like transporter on AM was evaluated using antipepT1 antisera and Western blot analysis. The substrate specificity of Arg-Gly and Arg-Gly-Asp was determined using purified inducible nitric oxide synthase (iNOS). The availability of ACPs in the lung was determined by the HPLC analysis of plasma and (BAL) fluid.;Results. The FITC-labeled peptides were internalized by AM without degradation. Uptake of Arg-Lys*, beta-Ala-Lys*, and Gly-Sar* was blocked (∼50%) by cephradine, but not by Lys (an inhibitor on CAT-2B for arginine transport). The NO production by AM through ACPs was significantly blocked by PepT1 inhibitors and by an antiPepT1 antibody in a dose-dependent manner. These inhibitors had no effect on AM production of NO using Arg as a substrate. Arg-Gly and Arg-Gly-Asp were found to be direct substrates for iNOS with similar Km and Vmax values to those of Arg. But the production of NO by AM using ACPs as substrate was 2-fold higher than using Arg as a substrate. Both Arg-Gly and Arg-Gly-Asp were found in rat plasma and BAL fluid. The presence of a PepT1-like transporter on AM was confirmed by Western blot.;Conclusion. This study shows that AM exhibits PepT1-like transporter for small peptide uptake. ACPs, through PepT1-like transporter, can serve as direct substrates for AM production of NO, an important mediator on both protection the lung from bacteria infection and augments inflammation lung injury.

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