Figure 1.
Genotype of mutant gene of Akita mouse by PCR analysis polymerase chain reaction (PCR) amplification was digested at 280 bp, while the control wild type (WT) gene at 140 bp (panel A). Differences in blood glucose levels (mg/dL, panel B), systolic blood pressure (SBP, panel C), and body weight (g, panel D) were observed between control WT mice and their time-course Akita diabetic mice. *p<0.05; ***p<0.001, compared with its WT group.
Figure 2.
Arginase activity/expression in the aorta.
Time-course analysis showed significant increases in arginase activity in diabetic mice (12 and 24 wks of age) as compared pre-diabetic mice (4 wks of age) (panel A). Pretreatment with (S)-(2 boronoethyl)-L-cysteine (BEC, 100 µmol/l, an arginase inhibitor) blocked this elevation. Arginase 1 (Arg1) expression in the Akita mice was slightly increased at 4 wks (panel B) and markedly increased at 12 (panel C) and 24 wks of age (panel D). Levels of arginase 2 (Arg2) were not different among age-matched WT and Akita mice (panel B–D). Values are expressed as mean ± S.E.M. of 4–5 independent experiments. * p<0.05; ** p<0.01; *** p<0.001compared to its WT group; # p<0.05, compared to Akita group.
Figure 3.
Arginase activity/expression in the corpus cavernosum (CC).
Time-course analysis of cavernosal arginase activity at pre-diabetic stage (4 wks of age) and diabetic (12 and 24 wks of age) stage (panel A). Pretreatment with (S)-(2 boronoethyl)-L-cysteine (BEC, 100 µmol/l, an arginase inhibitor) significantly prevented these increases. Levels of both arginase 1 (Arg1) and arginase 2 (Arg2) were markedly increased in pre-diabetic (panel B) and diabetic (panel C and D) Akita mice. Values are expressed as mean ± S.E.M. of 4–5 independent experiments. * p<0.05; ** p<0.01; *** p<0.001compared to its WT group; # p<0.05, compared to Akita group.
Figure 4.
Impairment of endothelium-dependent relaxation in the aorta.
Concentration-response curves to acetylcholine (ACh, 0.001–10 µmol/l) were analysed at 4 (panel A), 12 (panel B), and 24 wks of age (panel C) in aorta precontracted with phenylephrine (PE). The maximal efficacy (Emax) and potency (pEC50) values of ACh-induced aortic relaxation are presented in panels D and E. Data were calculated relative to the maximal changes from the contraction produced by PE (1 µmol/l), which was taken as 100%. Data are the means ± S.E.M. of 5–7 experiments. # p<0.05; ## p<0.01, indicates differences in pEC50 values of the dose-response curves; * p<0.05; ** p<0.01, compared with its WT mice group.
Figure 5.
Impairment of endothelium-dependent relaxation in the CC.
Concentration-response curves to acetylcholine (ACh, 0.001–3 µmol/l) were analysed at 4 (panel A), 12 (panel B), and 24 wks of age (panel C) in CC precontracted with phenylephrine (PE, 10 µmol/l). The maximal efficiency (Emax) and potency (pEC50) values of ACh-induced aortic relaxation of WT and Akita are presented in panels D and E. Data were calculated relative to the maximal changes from the contraction produced by PE, which was taken as 100%. Data are the means ± S.E.M. of 4–7 experiments. * p<0.05, compared with its WT mice group.# p<0.05, compared to WT mice at 4 wks of age.
Figure 6.
Reduced cavernosal nitrergic relaxation is enhanced by inhibition of arginase.
EFS-induced nitrergic relaxations (1–32 Hz) were performed at 4 (panel A), 12 and 24 weeks of age. Pretreatment with an arginase inhibitor ABH (100 µmol/l) significantly attenuated impaired nitrergic relaxation in Akita mice at 12 (panel B) and 24 (panel C) wks of age, whereas treatment with L-NAME (100 100 µmol/l) fully abolished EFS-induced nitrergic relaxation in CC of WT and Akita mice (panel D). Data represent the means ± S.E.M. of 5–6 experiments. * p<0.05 and ** p<0.01, compared with its respective frequency in WT mice; # p<0.05, compared with its respective frequency in Akita mice.
Figure 7.
Inhibition of Arginase enhances endothelium-dependent relaxation.
Relaxation was induced by a single dose of ACh (1 µmol/l) in aorta (panel A and B) and corpus cavernosum (CC, panel C and D) in the absence or presence of an arginase inhibitor, BEC or ABH (100 µmol/l each) in wild type (WT) and Akita mice. Concentration-response curve to ACh (0.001–10 µmol/l) in aorta of WT and Akita mice at 24 wks of age in the absence or presence of BEC (100 µmol/l) (panel E). Data represent the means ± S.E.M. of 4–6 experiments. * p<0.05 and ** p<0.01, compared with control WT mice; # p<0.05, compared with its respective concentration or time course in Akita mice.
Figure 8.
Protein levels of phospho-eNOSSer−1177 in aorta and corpus cavernosum (CC).
Levels of phospho eNOS (at Ser-1177), total eNOS, and β-actin were determined in aorta (panel A–C) and CC (panel D–F) from wild type (WT) littermate and Akita mice at 4, 12 and 24 weeks of age. Representative blots are shown in the top of each panel. Densitometric analysis was carried using Gene Snap software, results normalized to β-actin and expressed as arbitrary units. Data represent the mean ± S.E.M. of 6 experiments (each group). * p<0.05; ** p<0.01, compared to WT mice.
Figure 9.
Levels of nNOS protein and NOS activity in the corpus cavernosum (CC).
Protein levels of nNOS were analysed in non-diabetic and diabetic cavernosal tissues at 12 and 24 weeks of age. Representative blots of nNOS (panel A). Densitometric analysis was carried our using Gene Snap software, results normalized to β-actin and expressed as arbitrary units. NOS activity was determined by the conversion of L-arginine to L-citrulline in the absence and presence of the arginase inhibitor BEC (100 µmol/l) in aorta and CC from control WT and Akita mice (Panel B). Values are expressed as mean ± S.E.M. of 4 experiments per group. * p<0.05; *** p<0.001, compared to WT mice; # p<0.05, compared with its Akita control.