Endothelial nitric oxide synthase keeps erection regulatory function balance in the penis

Abstract

Objectives: We evaluated the regulatory influence of endothelial nitric oxide (NO) on the basal functional states of the NO and RhoA/Rho-kinase signaling pathways in the penis using endothelial NO synthase (eNOS) mutant mice and eNOS gene transfer technology.

Methods: Four groups of mice were used: wild type (WT), eNOS gene deleted (eNOS-/-), eNOS and neuronal NOS gene deleted (dNOS-/-), and eNOS-/- mutant mice transfected intracavernosally with eNOS. Cyclic guanosine monophosphate (cGMP) concentration, protein kinase G (PKG) activity, activated RhoA, and Rho-kinase activity were determined in penes of WT and both mutant mouse groups. Constitutive NOS and PKG activities, RhoA, Rho-kinase-alpha and -beta isoforms, and phosphorylated myosin light-chain phosphatase target subunit (p-MYPT-1) expressions and Rho-kinase activity were determined in penes of eNOS-/- mice after eNOS gene transfer.

Results: Compared with results in the WT penis, eNOS-/- and dNOS-/- mutant mouse penes had significant reductions in NOS activity, cGMP concentration, PKG activity, Rho-kinase activity, and p-MYPT-1 expression (p<0.05) with no significant changes in activated RhoA or in RhoA and Rho-kinase-alpha and -beta protein expressions. After eNOS gene transfer to penes of eNOS-/- mice, Rho-kinase-beta and p-MYPT-1 expressions and total Rho-kinase activity were significantly increased from baseline levels (p<0.05).

Conclusions: These data suggest that endothelial NO has a role in the penis as a regulator of the basal signaling functions of the NO and RhoA/Rho-kinase erection mediatory pathways. These data offer new insight into the homeostasis of erection regulatory biology.

Figure 1

(a) NOS activity as measured by calcium-dependent conversion of L-arginine to L-citrulline (b) cGMP level, and (c) PKG activity in WT, eNOS−/−, and dNOS−/− mouse penes. n indicates number of tissue samples; * (p<0.05) when compared to WT.

Figure 2

(a) RhoA activation and (b) Rho-kinase activity in WT, eNOS−/−, and dNOS−/− mouse penes. n indicates number of tissue samples; * (p<0.05) when compared to WT.

Figure 3

Western blot analysis demonstrating penile expression of (a) RhoA (membrane), (b) Rho-kinase-α (cytosol), (c) Rho-kinase-β (cytosol), and (d) p-MYPT-1 (cytosol) in WT (lane 1), dNOS−/− (lane 2), eNOS−/− (lane 3) mouse penes and eNOS−/− mouse penes after intracavernous administration of AdCMVeNOS (lane 4). Quantitative densitometry of the protein expression for RhoA, Rho-kinase-α, Rho-kinase-β, and p-MYPT-1 (ratio of targeted protein expressed per β-actin) is shown in each panel. n indicates number of tissue samples; ρ (p<0.05) when compared to WT, eNOS−/−, and dNOS−/−; * (p<0.05) when compared to WT; ** (p<0.05) when compared to eNOS−/− and dNOS−/−.

Figure 4

(a) NOS activity as measured by calcium-dependent conversion of L-arginine to L-citrulline and (b) PKG activity in WT, eNOS−/−, and dNOS−/− mouse penes and eNOS−/− mouse penes after intracavernous administration of AdCMVeNOS. n indicates number of tissue samples; * (p<0.05) when compared to WT; ** (p<0.05) when compared to eNOS−/− and dNOS−/−mice.

Figure 5

Rho-kinase activity in WT, eNOS−/−, and dNOS−/− mouse penes and eNOS−/− mouse penes after intracavernous administration of AdCMVeNOS. n indicates number of tissue samples; * (p<0.05) when compared to WT; ** (p<0.05) when compared to eNOS−/− and dNOS−/−mice.