Figure 3. eNOS controls the UPEC invasion of mouse bladder. (A) NOS and its product NO regulate the dynamin2-dependent bacterial invasion of BECs. Inhibition of NOS activity with 100 µM L-NAME significantly decreases the E. coli ORN103(pSH2) invasion of empty vector (EV)–transfected control BECs (open bars) as well as BECs overexpressing wild-type dynamin2 (shaded bars), and treatment with 500 µM S-nitrosothiol deta-NO increases the bacterial invasion of control (open bars) but not dynamin2-overexpressing BECs (shaded bars). Data shown are for n = 3; *, P < 0.05 versus untreated control empty vector–expressing BECs; **, P < 0.01 versus untreated control empty vector–transfected BECs; and ‡, P < 0.01 versus untreated wild-type dynamin2-overexpressing BECs (all by ANOVA). (B) Bacterial invasion after exposure of BECs expressing GFPshRNA or dynamin2shRNA/eNOSshRNA to the UPEC strains CFT073, UTI89, or isogenic control UTI89ΔFimH. For CFT073, data are shown relative to GFPshRNA-expressing cells. For UTI89 and UTI89ΔFimH, data are shown relative to GFPshRNA-expressing cells that were incubated with UTI89. n = 3; **, P < 0.05 versus values from corresponding GFPshRNA-transfected samples. (C) L-NAME attenuates the bacterial invasion of mouse bladder epithelium. Isolated strips of the bladder transitional epithelium layer were either incubated or not incubated with L-NAME and E. coli ORN103(pSH2) for 2 h followed by the determination of bacterial invasion using the gentamicin protection assay. Data for each treatment group were pooled from five animals; *, P < 0.05 versus untreated samples by ANOVA (NT, not treated). (D) eNOS−/− bladders exhibit a decreased invasiveness by UPEC. Bladders from eNOS−/− and isogenic control mice were harvested, and transitional epithelium layers were isolated and used for invasion by the UPEC UTI89. Bacterial invasion was assessed by gentamicin protection assay followed by colony counts. n = 4; *, P < 0.01 versus wild-type (WT) animals. CFU, colony-forming unit. (E) The level of dynamin2 protein expression is not changed in bladder epithelia obtained from eNOS knockout (eNOS−/−) compared with wild-type mice. Data in A–D represent means ± SEM.
Figure 3.

eNOS controls the UPEC invasion of mouse bladder. (A) NOS and its product NO regulate the dynamin2-dependent bacterial invasion of BECs. Inhibition of NOS activity with 100 µM L-NAME significantly decreases the E. coli ORN103(pSH2) invasion of empty vector (EV)–transfected control BECs (open bars) as well as BECs overexpressing wild-type dynamin2 (shaded bars), and treatment with 500 µM S-nitrosothiol deta-NO increases the bacterial invasion of control (open bars) but not dynamin2-overexpressing BECs (shaded bars). Data shown are for n = 3; *, P < 0.05 versus untreated control empty vector–expressing BECs; **, P < 0.01 versus untreated control empty vector–transfected BECs; and ‡, P < 0.01 versus untreated wild-type dynamin2-overexpressing BECs (all by ANOVA). (B) Bacterial invasion after exposure of BECs expressing GFPshRNA or dynamin2shRNA/eNOSshRNA to the UPEC strains CFT073, UTI89, or isogenic control UTI89ΔFimH. For CFT073, data are shown relative to GFPshRNA-expressing cells. For UTI89 and UTI89ΔFimH, data are shown relative to GFPshRNA-expressing cells that were incubated with UTI89. n = 3; **, P < 0.05 versus values from corresponding GFPshRNA-transfected samples. (C) L-NAME attenuates the bacterial invasion of mouse bladder epithelium. Isolated strips of the bladder transitional epithelium layer were either incubated or not incubated with L-NAME and E. coli ORN103(pSH2) for 2 h followed by the determination of bacterial invasion using the gentamicin protection assay. Data for each treatment group were pooled from five animals; *, P < 0.05 versus untreated samples by ANOVA (NT, not treated). (D) eNOS−/− bladders exhibit a decreased invasiveness by UPEC. Bladders from eNOS−/− and isogenic control mice were harvested, and transitional epithelium layers were isolated and used for invasion by the UPEC UTI89. Bacterial invasion was assessed by gentamicin protection assay followed by colony counts. n = 4; *, P < 0.01 versus wild-type (WT) animals. CFU, colony-forming unit. (E) The level of dynamin2 protein expression is not changed in bladder epithelia obtained from eNOS knockout (eNOS−/−) compared with wild-type mice. Data in A–D represent means ± SEM.

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