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Fig. 4. Western blot analysis and quantitative densitometry of the protein expressions of soluble guanylate cyclase in WT, nNOS^-/- (NOS1^-/-), eNOS^-/- (NOS3^-/-), and double mutant (NOS1/3^-/-) mouse penes. When normalized for b -actin protein expression, there was no significant difference between the four groups of mice.

Fig. 5. Biochemical activity measurements in WT and transgenic sickle cell mouse penes. (a) Ca²⁺-dependent NOS activity. (b) PDE5A enzyme activity. Both constitutive NOS activity and PDE5 activity were significantly reduced when compared to WT and SS+/- mice (*, P < 0.05). SS^-/- mice show a priapism phenotype, and this is associated with reductions of both NOS and PDE5A activities. SS+/- mice show a phenotype similar to that of WT. For all panels, data represent three observations done in duplicate for each group and are expressed as mean values ± SEM.

Fig. 6. PDE5A measurements in isolated WT mouse cavernosal strips after carbachol exposure. (a) Western blot analysis and quantitative densitometry of the protein expressions of the phosphorylated (active) form of PDE5A (p-PDE5A) and total PDE5A (t-PDE5A) under baseline conditions (C) and after exposure to the cholinergic analog carbachol (CCh) in the absence (10 min) and presence (20-90 min) of the NOS inhibitor l-NAME. CCh resulted in an increase in the expression of p-PDE5A while t-PDE5A was unaltered. Asterisk indicates P < 0.05 when compared to C (preCCh stimulation); double asterisk indicates P < 0.05 when compared to (10 min; exposure to CCh). (b) Western blot analysis and quantitative densitometry of the expression of sGC under baseline conditions (C) and after exposure to CCh in the absence (10 min) and presence (20-90 min) of the NOS inhibitor l-NAME. Unlike p-PDE5A expression, sGC protein expression did not change with CCh exposure or in the presence of L-NAME. For all panels, data represent four observations for each experiment and are expressed as mean values ± SEM.

Fig. 7. PDE5A measurements in isolated WT mouse cavernosal strips after NO exposure. Western blot analysis and quantitative densitometry of the protein expressions of the phosphorylated (activated) form of PDE5A (p-PDE5A) and total PDE5A (t-PDE5A) under baseline conditions (C) and after exposure to the NOS inhibitor l-NAME in the absence (30 min) and presence (60 and 90 min) of the NO donor DEA/NO. l-NAME treatment resulted in a decreased expression of p-PDE5A, whereas DEA/NO treatment resulted in its reversible increase. Levels of t-PDE5A were unaltered by these treatments. Asterisk indicates P < 0.05 when compared to C (pre-l-NAME); double asterisk indicates P < 0.05 when compared to 30 min exposure to l-NAME. For all panels, data represent five observations for each experiment and are expressed as mean values ± SEM.

Fig. 8. Effects of eNOS gene transfer in eNOS^-/- (NOS3^-/-) mice. (a) Ca²⁺-dependent and -independent NOS activity at baseline and after transfection with Adb gal or AdNOS3. Asterisk indicates P < 0.05 when compared to NOS3^-/- at baseline. (b) Absolute change in cGMP levels in response to maximal CNS in vivo at baseline and after transfection with Adb gal or AdNOS3. Asterisk indicates P < 0.05 when compared to WT; double asterisk indicates P < 0.05 when compared to NOS3^-/-. For all panels, data are expressed as mean values ± SEM.

*, P < 0.05 vs. WT.

nNOS, neuronal NOS; eNOS, endothelial NOS; n = 4-6

*, P < 0.05 vs. WT.